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Magnesium inthe CentralNervousSystem


Magnesium inthe CentralNervousSystem


EDITEDBY ROBERTVINK
DisciplineofAnatomyandPathology&AdelaideCentreforNeuroscienceResearch,SchoolofMedical Sciences,TheUniversityofAdelaide,Adelaide,SouthAustralia,Australia

MIHAINECHIFOR
DepartmentofPharmacology,Gr.T.PopaUniversityofMedicineandPharmacy,Iasi,Romania

PublishedinAdelaideby UniversityofAdelaidePress BarrSmithLibrary TheUniversityofAdelaide SouthAustralia 5005 press@adelaide.edu.au www.adelaide.edu.au/press TheUniversityofAdelaidePresspublishesexternallyrefereedscholarlybooksbystaffoftheUniversityof Adelaide.Itaimstomaximisetheaccessibilitytoitsbestresearchbypublishingworksthroughtheinternet asfreedownloadsandashighqualityprintedvolumesondemand. ElectronicIndex:thisbookisavailablefromthewebsiteasadownloadablePDFwithfullysearchabletext. Pleaseusetheelectronicversiontoserveastheindex. 2011TheAuthors Thisbookiscopyright.Apartfromanyfairdealingforthepurposesofprivatestudy,research,criticismor reviewaspermittedundertheCopyrightAct,nopartmaybereproduced,storedinaretrievalsystem,or transmitted,inanyformorbyanymeans,electronic,mechanical,photocopying,recordingorotherwise withoutthepriorwrittenpermission.AddressallinquiriestotheDirectorattheaboveaddress. ForthefullCataloguinginPublicationdatapleasecontactNationalLibraryofAustralia Magnesiuminthecentralnervoussystem/editedbyRobertVinkandMihaiNechifor. IVink,Robert. IINechifor,Mihai. 1MagnesiumPhysiologicaleffect. 2Magnesiuminthebody. 3Centralnervoussystem. ISBN(electronic)9780987073051 ISBN(paperback)9780987073068 Bookdesign:RobertVink Coverdesign:EmmaSpoehr.Photographwithpermission,istockphoto.com PaperbackprintedbyGriffinPress,SouthAustralia

Magnesium in the Central Nervous System

List of Contributors

Bulent Ahishali Istanbul University, Istanbul Faculty of Medicine, Department of Physiology & Department of Histology and Embryology, Capa 34093 Istanbul, Turkey Jean-Pierre Bali Universit Montpellier I, 34000 Montpellier, France Jean-Marie Billard Universit Paris Descartes, Facult de Mdecine Ren Descartes, Centre de Psychiatrie et Neurosciences, UMR 894, Paris, 75014, France Kym Campbell Centre for Neuromuscular and Neurological Disorders, University of Western Australia & Australian Neuromuscular Research Institute, Department of Neurosurgery, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia Yves Cazals UMR6231, Universit Paul Czanne, Marseille, France Zheng Chen Department of Psychiatry & Institute for Geriatric Clinic and Rehabilitation, Beijing Geriatric Hospital, Beijing 100095, China Dehua Chui Neuroscience Research Institute & Department of Neurobiology, Key Laboratory for Neuroscience, Ministry of Education & Ministry of Public Health, Health Science Center, Peking University, Beijing 100191, China Naomi L. Cook Discipline of Anatomy and Pathology, School of Medical Sciences & Adelaide Centre for Neuroscience Research, The University of Adelaide, Adelaide, South Australia, Australia Frances Corrigan Discipline of Anatomy and Pathology, School of Medical Sciences & Adelaide Centre for Neuroscience Research, The University of Adelaide, Adelaide, South Australia, Australia Magdalena D. Cuciureanu Department of Pharmacology, Gr. T. Popa University of Medicine and Pharmacy 16, Iasi, Romania Sang-Hwan Do Department of Anesthesiology and Pain medicine, Seoul National University Bundang Hospital, Seongnam, South Korea George A. Eby III George Eby Research Institute, 14909-C Fitzhugh Road, Austin, Texas, USA Karen L. Eby George Eby Research Institute, 14909-C Fitzhugh Road, Austin, Texas, USA Mounir N. Ghabriel Discipline of Anatomy and Pathology, School of Medical Sciences & Adelaide Centre for Neuroscience Research, The University of Adelaide, Adelaide, South Australia, Australia Tomoyo Hashimoto Department of Internal Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Nagano, Japan Michael R. Hoane Restorative Neuroscience Laboratory, Brain and Cognitive Science Program, Department of Psychology Southern Illinois University, Carbondale, Illinois, USA Xavier Holy Institut de Recherche Biomdicale des Armes, Antenne de Brtigny, Brtigny-sur-Orge, France Stefano Iotti Dipartimento di Medicina Interna, dellInvecchiamento e Malattie Nefrologiche, Universit di Bologna, Italy and Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy v

Magnesium in the Central Nervous System

Mehmet Kaya Istanbul University, Istanbul Faculty of Medicine, Department of Physiology & Department of Histology and Embryology, Capa 34093 Istanbul, Turkey Neville W. Knuckey Centre for Neuromuscular and Neurological Disorders, University of Western Australia, & Australian Neuromuscular Research Institute, Department of Neurosurgery, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia Marzia Leidi Universit di Milano, Dipartimento di Scienze Cliniche Luigi Sacco, Via G.B. Grassi 74, 20157 Milano, Italy Yi Liu Neuroscience Research Institute & Department of Neurobiology, Key Laboratory for Neuroscience, Ministry of Education & Ministry of Public Health, Health Science Center, Peking University, Beijing 100191, China Jeanette A.M. Maier Universit di Milano, Dipartimento di Scienze Cliniche Luigi Sacco, Via G.B. Grassi 74, 20157 Milano, Italy Emil Malucelli Dipartimento di Medicina Interna, dellInvecchiamento e Malattie Nefrologiche, Universit di Bologna, Italy Lucia Mastrototaro Istituto di Patologia Generale e Centro di Ricerche Oncologiche Giovanni XXIII, Universit Cattolica del Sacro Cuore, Rome, Italy Alexander Mauskop The New York Headache Center, 30 East 76th Street, New York, NY, USA Bruno P. Meloni Centre for Neuromuscular and Neurological Disorders, University of Western Australia, & Australian Neuromuscular Research Institute, Department of Neurosurgery, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia Marianne Mousain-Bosc Ecole de lADN, Museum dHistoire Naturelle de Nmes, 19, Grandrue, 30000 Nimes, France Harald Murck Clinic of Psychiatry and Psychotherapy, University of Marburg, Marburg, Germany Hyo-Seok Na Department of Anesthesiology and Pain medicine, Seoul National University Bundang Hospital, Seongnam, South Korea Mihai Nechifor Department of Pharmacology, Gr. T. Popa University of Medicine and Pharmacy, Iasi, Romania Kiyomitsu Oyanagi Department of Brain Disease Research, Shinshu University School of Medicine, Nagano Japan Victoria Papadopol Regional Centre of Public Health Iai, Romania Florent Raffin Institut de Recherche Biomdicale des Armes, Antenne de la Tronche, La Tronche, France Andrea M.P. Romani Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio USA Harry Rubin Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA, USA Jung-Hee Ryu Department of Anesthesiology and Pain medicine, Seoul National University Bundang Hospital, Seongnam, South Korea vi

Magnesium in the Central Nervous System

Jeffrey L. Saver UCLA Stroke Center, Geffen School of Medicine of the University of California, Los Angeles, CA, USA Isabelle Sendowski Institut de Recherche Biomdicale des Armes, Antenne de la Tronche, La Tronche, France Christian Siatka Ecole de lADN, Museum dHistoire Naturelle de Nmes, 19, grandrue, 30000 Nimes, France Yuetao Song Department of Psychiatry & Institute for Geriatric Clinic and Rehabilitation, Beijing Geriatric Hospital, Beijing 100095, China Sidney Starkman UCLA Stroke Center, Geffen School of Medicine of the University of California, Los Angeles, CA, USA Valentina Trapani Istituto di Patologia Generale e Centro di Ricerche Oncologiche Giovanni XXIII, Universit Cattolica del Sacro Cuore, Rome, Italy Walter M. van den Bergh Department of Intensive Care, Academic Medical Center, 1100 DD Amsterdam, The Netherlands Corinna van den Heuvel Discipline of Anatomy and Pathology, School of Medical Sciences & Adelaide Centre for Neuroscience Research, The University of Adelaide, Adelaide, South Australia, Australia Robert Vink Discipline of Anatomy and Pathology, School of Medical Sciences & Adelaide Centre for Neuroscience Research, The University of Adelaide, Adelaide, South Australia, Australia Weishan Wang Department of Psychiatry & Institute for Geriatric Clinic and Rehabilitation, Beijing Geriatric Hospital, Beijing 100095, China Federica I. Wolf Istituto di Patologia Generale e Centro di Ricerche Oncologiche Giovanni XXIII, Universit Cattolica del Sacro Cuore, Rome, Italy Lisa A. Yablon The New York Headache Center, 30 East 76th Street, New York, NY, USA Huan Yang Neuroscience Research Institute & Department of Neurobiology, Key Laboratory for Neuroscience, Ministry of Education & Ministry of Public Health, Health Science Center, Peking University, Beijing 100191, China Jia Yu Department of Psychiatry & Institute for Geriatric Clinic and Rehabilitation, Beijing Geriatric Hospital, Beijing 100095, China Honglin Zhang Department of Psychiatry & Institute for Geriatric Clinic and Rehabilitation, Beijing Geriatric Hospital, Beijing 100095, China

vii

Magnesium in the Central Nervous System

Preface
The brain is the most complex organ in our body. Indeed, it is perhaps the most complex structure we have ever encountered in nature. Both structurally and functionally, there are many peculiarities that differentiate the brain from all other organs. The brain is our connection to the world around us and by governing nervous system and higher function, any disturbance induces severe neurological and psychiatric disorders that can have a devastating effect on quality of life. Our understanding of the physiology and biochemistry of the brain has improved dramatically in the last two decades. In particular, the critical role of cations, including magnesium, has become evident, even if incompletely understood at a mechanistic level. The exact role and regulation of magnesium, in particular, remains elusive, largely because intracellular levels are so difficult to quantify routinely. Nonetheless, the importance of magnesium to normal central nervous system activity is self-evident given the complicated homeostatic mechanisms that maintain the concentration of this cation within strict limits essential for normal physiology and metabolism. There is also considerable accumulating evidence to suggest that alterations to some brain functions in both normal and pathological conditions may be linked to alterations in local magnesium concentration. This book, containing chapters written by some of the foremost experts in the field of magnesium research, brings together the latest in experimental and clinical magnesium research as it relates to the central nervous system. It offers a complete and updated view of magnesiums involvement in central nervous system function and in so doing, brings together two main pillars of contemporary neuroscience research, namely providing an explanation for the molecular mechanisms involved in brain function, and emphasizing the connections between the molecular changes and behaviour. It is the untiring efforts of those magnesium researchers who have dedicated their lives to unravelling the mysteries of magnesiums role in biological systems that has inspired the collation of this volume of work. Robert Vink Mihai Nechifor

viii

Magnesium in the Central Nervous System

List of Contributors Preface Section 1: Magnesium in Normal Brain Chapter 1 Free magnesium concentration in human brain Stefano Iotti and Emil Malucelli

v viii

3 13 59 75

Chapter 2 Chapter 3 Chapter 4 Chapter 5

Intracellular magnesium homeostasis Andrea M.P. Romani Magnesium transport across the blood-brain barriers Mounir N. Ghabriel and Robert Vink Intracellular free Mg2+ and MgATP2- in coordinate control of protein synthesis and cell proliferation Harry Rubin Magnesium and the Yin-Yang interplay in apoptosis Valentina Trapani, Lucia Mastrototaro and Federica I. Wolf

85 99

Chapter 6 Brain magnesium homeostasis as a target for reducing cognitive ageing Jean-Marie Billard Section 2: Magnesium in Neurological Diseases Chapter 7 The role of magnesium therapy in learning and memory Michael R. Hoane

115 125 135 145 157 167 181 193 205 217 229

Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17

The role of magnesium in headache and migraine Lisa A. Yablon and Alexander Mauskop Magnesium in edema and blood-brain barrier disruption Mehmet Kaya and Bulent Ahishali Magnesium and hearing loss Isabelle Sendowski, Xavier Holy, Florent Raffin and Yves Cazals The role of magnesium in pain Hyo-Seok Na, Jung-Hee Ryu and Sang-Hwan Do The role of magnesium in traumatic CNS injury Naomi L. Cook, Frances Corrigan and Corinna van den Heuvel The use of magnesium in experimental cerebral ischaemia Bruno P. Meloni, Kym Campbell and Neville W. Knuckey Magnesium in subarachnoid hemorrhage Walter M. van den Bergh Magnesium in clinical stroke Jeffrey L. Saver and Sidney Starkman Magnesium in cancer: more questions than answers Marzia Leidi, Federica I. Wolf and Jeanette A.M. Maier Magnesium in Parkinsons disease: an update in clinical and basic aspects Kiyomitsu Oyanagi and Tomoyo Hashimoto

Magnesium in the Central Nervous System

Section 3: Involvement of Magnesium in Psychiatric Diseases Chapter 18 Magnesium and Alzheimers disease Dehua Chui, Zheng Chen, Jia Yu, Honglin Zhang, Weishan Wang Yuetao Song, Huan Yang and Yi Liu

239

Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Chapter 24

Magnesium and stress Magdalena D. Cuciureanu and Robert Vink Magnesium in neuroses Victoria Papadopol and Mihai Nechifor Magnesium, hyperactivity and autism in children Marianne Mousain-Bosc, Christian Siatka and Jean-Pierre Bali Magnesium in psychoses (schizophrenia, bipolar disorder) Mihai Nechifor Magnesium and major depression George A. Eby III, Karen L. Eby and Harald Murck Magnesium in drug abuse and addiction Mihai Nechifor

251 269 283 303 313 331

Electronic Index: this book is available from the website adelaide.edu.au/press as a free down- loadable PDF with fully searchable text. Please use the electronic version to serve as the index.

Section 1 Magnesium in Normal Brain



Magnesium in the Central Nervous System

Chapter 1

Free magnesium concentration in the human brain


Stefano Iotti 1,2,and Emil Malucelli 1
1 2

stefano.iotti@unibo.it

Dipartimento di Medicina Interna, dellInvecchiamento e Malattie Nefrologiche, Universit di Bologna, Italy. Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy.

Abstract The cytosolic free magnesium concentration can be assessed in vivo in the human brain by phosphorus magnetic resonance spectroscopy. This technique has been employed in the human brain, providing new hints on the Mg2+ homeostasis and on its involvement on the cellular bioenergetics. The free cytosolic [Mg2+] measured in the human brain is about half of that assessed in the human skeletal muscle. This result is likely related to the lower ATP concentration of brain tissue. The possibility to assess the cytosolic [Mg2+] in the human brain opened the chance to study the involvement of Mg2+ in different neurological pathologies, and particularly in those where the defective mitochondrial energy production represents the primary causative factor in pathogenesis. The results obtained, studying patients affected by different types of mitochondrial cytopathies, helped to clarify the functional relationship between the energy metabolism and free [Mg2+], providing evidence that the cytosolic [Mg2+] is a function of the energy charge of brain cells and a defective mitochondrial respiration causes a derangement of cytosolic [Mg2+] homeostasis. A reduced cytosolic [Mg2+] has been also found in the occipital lobes of patients with different types of migraine and cluster headache, showing among migraine patients a trend in keeping with the severity of clinical phenotype. In addition, the assessment by phosphorus magnetic resonance spectroscopy of brain [Mg2+] can help in the differential diagnosis of Multiple System Atrophy and Parkinson's disease, offering a new diagnostic tool that may help to differentiate neurodegenerative diseases sharing common clinical features.

Introduction Inorganic phosphate (Pi), phosphocreatine (PCr), and ATP are the principal Mg+2 ligands among the phosphorylated molecules present in the cell cytosol detectable by phosphorus magnetic resonance spectroscopy (31P-MRS) (Figure 1). Nevertheless, in the cytosol matrix, magnesium is primarily bound to ATP. As a consequence, in the living organism, ATP is present mainly in the form MgATP2- (Iotti et al., 1996) (Figure 2) that is the active species in enzyme binding (Kuby and Noltman, 1959), as well as the energy producing form in active transport (Skou, 1982; Repke, 1982) and muscular contraction (Ramirez and Marecek 1980; Wells et al., 1980). The amount of magnesium bound to ATP shifts the resonance frequencies measured by 31P-MRS of signals coming from the three phosphoric groups , , of the molecule (chemical shift). Due to the chemical equilibrium between the Mg bound to ATP and free Mg2+, the chemical shift of ATP signals is function of free Mg2+ concentration

(Figure 3). The in vivo assessment of cytosolic Mg2+ concentration ([Mg2+]) in the human brain needs a specific calibration curve that should satisfy precise criteria. In particular, the calibration curve should take into account: i) other ions present in the cell cytosol competing with Mg2+ in binding ATP such as H+, Na+, K+, and ii) other ligands such as ADP, PCr and Pi competing with ATP in binding Mg2+ (Iotti et al., 1996). Operatively, [Mg2+] is assessed by 31P-MRS from the chemical shift of -ATP being measured from the signal of either -ATP (Gupta et al., 1984; Williams and Smith, 1995) or PCr (Taylor et al., 1991; Halvorson et al., 1992). The reliability of the in vivo measurements depends on the availability of an appropriate in vitro calibration method to determine the limits of chemical shifts of unbound ATP and Mg-ATP complexes. As a result, the calibration method has to take into account the two issues mentioned above together with other variables such as the ionic strength, temperature and pH of the medium. Besides, to have a precise measurement of -ATP 3

Human brain [Mg ]

2+

Chapter 1

Figure 1. Scheme of the main phosphorylated molecules present in the brain cell cytosol detectable by phosphorus magnetic resonance spectroscopy. Inorganic phosphate (Pi), phosphocreatine (PCr), and ATP are the principal magnesium ligands. chemical shift it must be taken into account that Building a calibration curve for the [Mg2+] the shifts of both -ATP and PCr change as a determination in human brain by 31P MRS 2+ function of [Mg ] (Iotti et al., 1996). The earlier The accuracy and reliability of in vivo assessment works of Gupta (Gupta et al., 1978; Gupta et al., of [Mg2+] mainly depend on the availability of 1983; Gupta et al., 1984) were based on the calibration curves based on precise in vitro NMR assumption of the presence of only one ATP-Mg measurements performed on appropriate complex. In the 1990s several studies were solutions that mimic, as far as possible, the in vivo published proposing more accurate calibration conditions of the tissue to be studied. As a methods taking into account equilibria involving consequence, these solutions will contain few different chemical species (Mosher et al., molecules involved in multiple equilibria in which 1992; Halvorson et al., 1992; Golding and several ligands (Lewis base) interact with metal Golding, 1995). Nevertheless, the influence of the ions (Lewis acid), and among them Mg2+ and H+. ionic strength (I) of the medium on the Typically, to build a calibration curve, a titration equilibrium constants of the species present in of the species of interest must be performed, the calibration solutions must be taken into linking its concentration to the experimental account as discussed in one report published in NMR measurement. [Mg2+] can be assessed by those years (Mottet et al., 1994). 31 P MRS from the chemical shift of -ATP signal, In the next section the methodological approach which in turn depends on the fraction of total used to build the calibration curve for the [Mg2+] ATP bound to Mg2+. Quite problematic is the use 31 determination in human brain by P-MRS is of a Mg electrode to measure [Mg2+] in a complex analytically presented. 4

Magnesium in the Central Nervous System

Chapter 1

Figure 2. Concentration of the major ATP- containing species present in solutions mimicking the cell brain cytosol plotted versus pMg. Solutions composition were: ATP = 3 mM, phos- phocreatine = 4.5 mM, K2HPO4 = 1.0 mM, and variable amounts of MgCl2 at pH = 7.00, 37 C and ionic strength 0.25 M. Adapted from Iotti et al., 1996. mixture, having to deal with interferences and lack of accuracy. A convenient approach to measure the [Mg2+] in a multi-equilibrium system is using appropriate algorithms and software, which are currently available, allowing a quantitative chemical speciation of any component present in solution, given a set of stability and acidic dissociation constants. In this approach, a pivotal step is the definition of the chemical model better describing the multi-equilibrium system present in solution. After the characterization of ligands and Lewis acids as the basic species, the next step is the delineation of all the possible equilibria between them. In the calibration curve specifically developed for the in vivo assessment of [Mg2+] in human brain (Iotti et al., 1996) the chemical model was defined by choosing four ligandsATP4-, PO43-, Cl- and PCr2- and four Lewis acidsNa+, K+, Mg2+ and H+ as basic species, giving raise to the multi- equilibrium system shown in Figure 4. An obligatory prerequisite for the analysis of spectra recorded in vitro is a reliable and homogeneous set of equilibrium constants to be used in the quantitative speciation of the experi- mental solutions. Therefore, if the equilibrium

Figure 3. Top: Sagittal slice image of human brain acquired by magnetic resonance imaging. The brain volume within vertical axes defines the region of occipital lobes. Bottom: 31P-MRS spectra acquired from occipital lobes placing the surface coil directly on the skull. Abbreviations: Pi, inorganic phosphate; PCr, phosphocreatine; , , , ATP phosphoric groups; PME, phospho- monoesters; PDE, phosphodiesters. Brain cytosolic [Mg2+] is assessed from the chemical shift of - ATP. constants reported in the literature do not conform to the conditions of temperature and ionic strength used in the experiments, suitable corrections, using Van't Hoff and/or Davies equations, have to be applied to account for the different conditions of NMR experiments (Iotti et al., 1996). Furthermore, the literature is rich with data pertaining to interactions between metal ions and ligands in solution, but the reported values are often characterized by extremely variable levels of precision and accuracy. Williams and colleagues (Duffield et al., 1991) discussed the problems related to the determination of the magnitude of quantities of biological/environ- 5

Human brain [Mg ]

2+

Chapter 1

Figure 4. Scheme of the chemical model used to build the calibration curve for the assessment of brain cytosolic [Mg2+] by 31P-MRS. Four ligands (ATP4-, PO43-, Cl-, and PCr2-) and four Lewis acids (Na+, K+, Mg2+, and H+) were chosen as basic species. mental interest and to its associated error. When these quantities are used for the chemical speciation of natural systems, the results bear an uncertainty directly related to the errors which affect the original quantities. Hence, a careful examination of the published values of equilibrium constants for the reactions involved in the chemical model chosen, must be performed. Another consideration to bear in mind is when using protonation constants that have been determined with the so-called "incomplete convention" (Halvorson et al., 1992), in which hydrogen ion activity is used instead of hydrogen ion concentration in the expression used to define the equilibrium constants. This can generate confusion when such constants are 6

used together with others that are expressed solely in terms of concentration. Therefore, a coherent and internally consistent set of equilibrium constants has to be collected, which are valid at 37 C and at I = 0.25 M, the conditions of solutions that mimic the in vivo environment. To handle the relevant number of different equilibria taken into consideration, an algorithm was developed that allowed a quantitative chemical speciation of the Mg2+-binding molecules with the goal of building a semi- empirical equation that correlates the chemical shift of the -ATP signal measured by in vitro NMR measurements in the calibration solutions to [Mg2+] calculated, taking into account the amount of Mg2+ bound to all constituents in solution. As a result, the concentrations of all species present under a given set of conditions (i.e. total concentrations of all reagents) were determined by the program HYSS (Alderighi et al., 1999), a specific software program, utilized for chemical speciation. Using this software it was possible to derive the concentration of free magnesium in any condition. HYSS requires two sets of data: a chemical model and the experimental conditions. The model was the one reported in Figure 4, by specifying values for the equilibrium constants abc.... and the corresp- onding stoichiometric coefficients a, b, c, ...., considering that the general reaction between reagents A, B, C ... to give a chemical species of formula AaBbCc: aA+bB+cC+ AaBbCc is associated with an equilibrium constant abc...: [AaBbCc...] abc = a b c [A] [B] [C] Electric charges have been omitted for the sake of simplicity. By using this approach and different calibration solutions prepared to mimic the in vivo metabolic conditions of brain, it has been possible to obtain, following an heuristic procedure, the following equation: pMg = x1 - log10 [( d - x2) 3/(x4 - d) 5] where x1, x2, x3, x4, x5 are the calculated parameters, is the measured chemical shift, and pMg = -log10 [Mg2+]. This equation can be seen as
X X

Magnesium in the Central Nervous System

Chapter 1

a more general expression of the typical titration curve in which x1 is the pK of the equilibrium involved and with x3, x5 = 1. More precisely, two different equations were obtained, where pMg is expressed as a function of the chemical shifts of -ATP from PCr () or from -ATP () (Iotti et al., 1996). However, the calibration curve obtained using as experimental variable showed to be more accurate than that obtained using (Iotti et al., 1996). This is because the measurements of in spectra obtained in vivo are usually less accurate than those of , due to the unresolved resonances of -ADP, NAD, NADH underlying up- field the -ATP peak. Furthermore, PCr resonates alone and is symmetric, allowing a more accurate identification of the centroid of the peak. Therefore, should be preferable to as experimental variable for the in vivo measure- ments of brain cytosolic free [Mg2+]. According to this, the equation to be used is:

pMg = 4.24 log10[( - 18.58)0.42 /(15.74 - )0.84] The specific software package MAGIC-BC based on the equation reported above has been made available at http://www.cermiv.unibo.it. It is worth underlying that, in principle, by this approach it is possible to assess the concen- tration of any species independently of the degree of complexity of the system under investigation, with the restriction of only two requisites: i) a precise definition of the chemical model and ii) the knowledge of the equilibrium constants of the chemical reactions involved. Cytosolic [Mg2+] in human brain The cytosolic [Mg2+] assessed by 31P-MRS in the occipital lobes of human brain of 36 control subjects (22 men and 14 women) of mean age 36.4 17.1 (SD) (16 - 67 years) was 0.182 mM (Iotti et al., 1996). Brain studies were performed on occipital lobes by placing the surface coil directly on the skull in the occipital region and precisely positioned by imaging the brain as shown in Figure 3. The free cytosolic [Mg2+] measured in the human brain is about half of that assessed in the human calf muscle (Iotti et al., 2000; Iotti and Malucelli, 2008). This result is likely related to the lower ATP concentration of brain tissue (3 mM) compared to that of skeletal muscle (8 mM), ATP being the major binding site present in the cellular milieu. Nevertheless, the

value found of 0.182 mM is lower than that obtained by others (Taylor et al., 1991; Halvorson et al., 1992). This is not a surprising result as the model considered in building this calibration curve contains more species that bind Mg2+ than in all other approaches published. As a conseq- uence, a lower value of cytosolic free [Mg2+] has to be expected. If the model described previously, upon which the cytosolic [Mg2+] assessment is based, had taken into account more species binding Mg2+, the resulting value of brain [Mg2+] would be even lower. Therefore, we believe that the present brain [Mg2+] value is a good estimate of the cytosolic free ion concentration, which however could still be slightly overestimated. In the cohort of subjects studied, there was no significant difference in cytosolic [Mg2+] as a function of age and sex. This shows that the sum of anions binding Mg2+in vivo does not change in the brain cortex during the life span studied and/or that regulatory mechanisms exist to maintain the cytosolic free [Mg2+] constant. The possibility to assess the free cytosolic [Mg2+] in the human brain opened the chance to study the involvement of Mg in different neurological pathologies. Particularly interesting are mito- chondrial cytopathies and migraines in which the defective mitochondrial energy production is respectively the primary causative or putative pathogenetic factor. Brain [Mg2+] in mitochondrial cytopathies 31 P-MRS was used to assess the free [Mg2+] in the occipital lobes of patients affected by different types of mitochondrial cytopathies due to known enzyme and/or mitochondrial DNA defects to clarify the functional relationship between the energy metabolism and the concentration of cytosolic free magnesium (Barbiroli et al., 1999a). In particular, 19 subjects (9 women and 10 men) of mean age 43 19 (SD) (13-75 years) with mitochondrial cytopathies were studied (see Table 1). Treatment with oral CoQ10 (150 mg/day) was given for six months to 9 patients. All patients displayed significantly low cytosolic [Mg2+], w ell below the 95% confidence interval of control values (Figure 5). Since the mtDNA mutations and enzyme defects found in these patients (Table 1) are known to be primarily responsible for brain defective mitochondrial respiration, this outcome strongly suggests that 7

Human brain [Mg ]

2+

Chapter 1

Table 1. Patients with mitochondrial cytopathies due to known enzyme/mtDNA defect/s. COX = cytochrome oxidase. Patients 1-4, 10, 12-14 and 16 were treated with CoQ10 (150 mg/day for six months). Adapted from Barbiroli et al., 1999a. Patients Sex/age (years) Diagnosis Enzyme defect or mtDNA bp numbers mutation/deletion 1* 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
* * * * * * * *

F/61 M/75 F/69 M/66 F/21 M/26 M/33 F/61 F/54 M/33 M/51 M/56 M/36 M/25 F/41 F/13 F/16 M/49 F/38

CPEO CPEO CPEO CPEO LHON LHON LHON LHON LHON LHON LHON LHON LHON LHON NARP NARP MERFF MELAS MEM

COX COX mtDNA deletion COX 3460 11778 11778 11778 11778 3460 11778 + 4216 + 13708 11778 11778 11778 8993 8993 8344 3243 mtDNA deletion

Abbreviations: * treated with CoQ10; CPEO, chronic progressive external ophthalmoplegia; LHON, Leber's hereditary optic neuropathy; NARP, neuropathy ataxia and retinitis pigmentosa; MEM, mitochondrial encephalomyopathy; MERF, myoclonic epilepsy with ragged red fibres; MELAS, mitochondrial myopathy encephalopathy, lactic acidosis, and stroke. the low cytosolic [Mg2+] found in these patients According to this view, the cytosolic [Mg2+] is a was secondary to failure of the respiratory chain. function of the energy charge of brain cells and a defective mitochondrial respiration causes a Nine of the 19 patients investigated were treated derangement of cytosolic [Mg2+] homeostasis. with CoQ, which improved the efficiency of the This interpretation is consistent with the respiratory chain (Barbiroli et al., 1999a). Admin- observations of several reports that indicate istration of CoQ also increased cytosolic [Mg2+] in mitochondria as the primary pool for the increase and decrease in cellular magnesium content, all treated patients (Figure 5). The increased suggesting that mitochondria might act as efficiency of oxidative phosphorylation following magnesium stores (Kubota et al., 2005; Farruggia treatment with CoQ accompanied by an et al., 2006) and play a key role in regulating increased cytosolic free [Mg2+] gives further magnesium homeostasis (Fatholahi et al., 2000; support to the hypothesis that changes in brain Murphy, 2000). cytosolic [Mg2+] were secondary to the improved efficiency of mitochondrial energy production. 8

Magnesium in the Central Nervous System

Chapter 1

Figure 5. Brain cytosolic [Mg2+] assessed in the occipital lobes of patients with mitochondrial cytopathies (open symbols). The values from nine of these patients after treatment with CoQ are shown by closed symbols. Dashed areas represent the 95% confidence intervals of 36 healthy control subjects. Adapted from Barbiroli et al., 1999a. Brain [Mg2+] in different types of migraine and cluster headache Low cytosolic free magnesium has been found in the brains of patients with migraine with and without aura during attacks (Ramadan et al., 1989) and interictally in young patients with migraine with aura (Lodi et al., 1997). It has been suggested that low free magnesium may contribute to the bioenergetic deficit in headache patients as magnesium is essential for mito- chondrial membrane stability and coupling of oxidative phosphorylation (Welch and Ramadan, 1995). Migraine headache is a common feature in patients with mitochondrial encephalomyopathies where deficient brain mitochondrial oxidation is due to mutations of mitochondrial DNA. In fact, several studies contributed to disclose an altered energy metabolism in the brain of patients with different types of migraine and cluster headache (Barbiroli et al., 1992; Montagna et al., 1994; Montagna et al., 1997), although the molecular mechanisms leading to oxidative deficit in migraine and cluster headache are unknown. On the other hand, as shown in the previous section, the reduction in cytosolic [Mg2+] seems to be secondary to the bioenergetics deficit in the brains of patients with different mitochondrial encephalomyopathies. These outcomes provided the rationale to perform an extensive study to

assess the brain Mg2+ by 31P-MRS in different form of migraines and in cluster headache (Lodi et al., 2001). The study was performed in 78 patients with different forms of migraine in attack-free periods (7 with migraine stroke, 13 with migraine with prolonged aura, 37 with migraine with typical aura or basilar migraine, 21 with migraine without aura), and 13 patients with cluster headache. In the occipital lobes of all subgroups of migraine and in cluster headache patients cytosolic [Mg2+] was significantly reduced. Among migraine patients the level of cytosolic free [Mg2+] correlated with the severity of clinical phenotype, showing the lowest values in patients with migraine stroke and the highest in patients with migraine without aura, as shown in Figure 6 (Lodi et al., 2001). Again, the results of this study sustain the hypothesis that the reduction of cytosolic [Mg2+] in tissues with defective mitochondrial functionality is secondary to the bioenergetics deficit, hence the reduction of brain cytosolic free [Mg2+] is unlikely to be a direct consequence of hypomagnesemia.

Figure 6. Concentration of cytosolic [Mg2+] in the occipital lobes of patients with migraine and cluster headache compared with healthy control subjects. Abbreviations: MS, migraine stroke; MwPA, migraine with prolonged aura; MwA, migraine with typical aura or basilar migraine; MwoA, migraine without aura; CH, cluster headache. All values are reported as mean SE. Adapted from Lodi et al., 2001. 9

Human brain [Mg ]

2+

Chapter 1

Brain [Mg2+] in the differential diagnosis of multiple system atrophy and idiopathic Parkinsons disease An in vivo study by 31P-MRS showed that the combined measurement of [PCr] and free [Mg2+] could help to differentiate patients with Multiple System Atrophy (MSA) from those with Parkinson's disease (PD) (Barbiroli et al., 1999b). MSA is a group of multisystem degenerative diseases that have several clinical features of PD. Furthermore, not all the signs in MSA become evident at the same time, rendering difficult the differentiation from PD. Therefore, differentiating MSA from PD can be complicated and the diagnosis of MSA represents a clinical challenge. The study was carried out on the occipital lobes of 15 patients with MSA and 13 patients with idiopathic PD. In the occipital lobes of MSA patients we found a 31 P-MRS spectroscopy pattern suggestive of defective brain mitochondrial respiration with low [PCr] and high [Pi]. However, the brain cytosolic [Mg2+] of MSA patients did not differ significantly from control values, displaying a peculiar pattern compared to mitochondrial cytopathies. On the other hand, PD patients did not exhibit in their occipital lobes the metabolic pattern typical of mitochondrial malfunction, showing an increased [Pi], decreased cytosolic [Mg2+] and unchanged [PCr] and pH. Although there is a highly significant difference in both [PCr] and [Mg2+] between MSA and PD patients when compared as groups, there is a small overlap of [PCr] and [Mg2+] values between the two groups when considering single individuals (Barbiroli et al., 1999b). However, taking into consideration both variables at the same time, the individual patients with MSA can be separated from the individual patients w ith PD References
Alderighi L, Gans P, Ienco A, Peters D, Sabatini A, Vacca A (1999) Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species. Coord Chem Rev 184:311-8.

Figure 7. Plot of the distribution patterns of 13 patients with Idiopathic Parkinsons Disease (closed symbols) and 15 patients with Multiple System Atrophy (open symbols) as a function of brain [PCr] and free [Mg2+] assessed by 31P-MRS. [PCr] and [Mg2+] alone were able to classify respectively 75% and 85% of the patients. Discriminant analysis using contemporarily these two independent parameters correctly classified 93% of cases. Adapted from Barbiroli et al., 1999b. with a probability of 93%, the degree of uncert- ainty being due to two cases (one case in each group, Figure 7). The results of the study revealed an abnormal bioenergetics in MSA and a decreased cytosolic [Mg2+] content in PD, offering a new diagnostic tool that may help to differ- entiate MSA from PD.

Acknowledgments This work was supported by an RFO grants from the University of Bologna and PRIN 2007ZT39FN from MIUR to Stefano Iotti.
Barbiroli B, Montagna P, Funicello R, Iotti S, Monari L, Pierangeli G, Zaniol P, Lugaresi E (1992) Abnormal 31 brain and muscle energy metabolism shown by P magnetic resonance spectroscopy in patients affected by migraine with aura. Neurology 42:1209-14.

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Magnesium in the Central Nervous System

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Barbiroli B, Iotti S, Cortelli P, Martinelli P, Lodi R, Carelli V, Montagna P (1999a) Low brain intracellular free magnesium in mitochondrial cytopathies. J Cereb Blood Flow Metab 19:528-32. Barbiroli B, Martinelli P, Patuelli A, Lodi R, Iotti S, Cortelli P, Montagna P (1999b) Phosphorus magnetic resonance spectroscopy in multiple system atrophy and Parkinson's disease. Mov Disord 14:430-5. Duffield JR, Marsicano F, Williams D R (1991) Chemical speciation modelling and thermodynamic database compilationI. Data uncertainties. Polyhedron 10:1105-11. Farruggia G, Iotti S, Prodi L, Montalti M, Zaccheroni N, Savage PB, Trapani V, Sale P, Wolf FI (2006) 8- Hydroxyquinoline derivatives as fluorescent sensors for magnesium in living cells. J Am Chem Soc 128:344- 50. Fatholahi M, LaNoue K, Romani A, Scarpa A (2000) 2+ Relationship between total and free cellular Mg( ) during metabolic stimulation of rat cardiac myocytes and perfused hearts. Arch Biochem Biophys 374:395- 401. Golding EM, Golding RM (1995) Interpretation of P MRS spectra in determining intracellular free magnesium and potassium ion concentration. Magn Reson Med. 33:467-74. Gupta RK, Benovic JL, Rose Z B (1978) The determination of the free magnesium level in the 31 human red blood cell by P NMR. J Biol Chem 252:6172-6. Gupta RK, Gupta P, Yushok WD, Rose ZB (1983) Measurements of the dissociation constant of MgATP at physiological nucleotide levels by a combination of 31 P NMR and optical absorbance spectroscopy. Biochem Biophys Res Commun 117:210-6. Gupta RK, Gupta P, Moore RD (1984) NMR studies of intracellular metal ions in intact cells and tissues. Ann Rev Biophys Bioeng 13:221-46. Halvorson HR, Vande Linde AMQ, Helpern JA, Welch KMA (1992) Assessment of magnesium concentration 31 by P NMR in vivo. NMR Biomed 5:53-8. Iotti S, Frassineti C, Alderighi L, Sabatini A, Vacca A, Barbiroli B (1996) In vivo assessment of free 31 magnesium concentration in human brain by P-MRS. A new calibration curve based on a mathematical algorithm. NMR Biomed 9:24-32.
31

Iotti S, Frassineti C, Alderighi L, Sabatini A, Vacca A, 31 Barbiroli B (2000) In vivo P-MRS assessment of 2+ cytosolic [Mg( )] in the human skeletal muscle in different metabolic conditions. Magn Reson Imag 18:607-14. Iotti S, Malucelli E (2008) In vivo assessment of Mg in 31 human brain and skeletal muscle by P-MRS. Magnesium Research 21:157-62. Kubota T, Shindo Y, Tokuno K, Komatsu H, Ogawa H, Kudo S, Kitamura Y, Suzuki K, Oka K (2005) Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells. Biochim Biophys Acta 1744(1):19-28. Kuby SA, Noltman EA (1959) ATP-Creatine Transphosphorylase, in: P.D. In: The Enzymes, (Boyer Ed), 2nd ed, New York, PA: Academic Press, 515603. Lodi R, Montagna P, Soriani S, Iotti S, Arnaldi C, Cortelli P et al. (1997) Deficit of brain and skeletal muscle bioenergetics and low brain magnesium in juvenile 31 migraine: an in vivo P magnetic resonance spectroscopy interictal study. Pediatr Res 42:866-71. Lodi R, Iotti S, Cortelli P, Pierangeli G, Cevoli S, Clementi V, Soriani S, Montagna P, Barbiroli B (2001) Deficient energy metabolism is associated with low free magnesium in the brains of patients with migraine and cluster headache. Brain Res Bull 54:437-41. Montagna P, Cortelli P, Monari L, Pierangeli G, Parchi P, Lodi R, Iotti S, Frassineti C, Zaniol P, Lugaresi E 31 (1994) P-magnetic resonance spectroscopy in migraine without aura. Neurology 44:666-9. Montagna P, Lodi R, Cortelli P, Pierangeli G, Iotti S, Cevoli S, Zaniol P, Barbiroli B (1997) Phosphorus magnetic resonance spectroscopy in cluster headache. Neurology 48:113-8. Mosher TJ, Williams GD, Doumen C, LaNoue KF, Smith MB (1992) Errors in the calibration of MgATP chemical shift limit: effects on the determination of free 31 magnesium by P NMR spectroscopy. Magn Reson Med 24:163-9. Mottet I, Demeure R, Gallez B, Grandin C, Van Beers 31 BE, Pringot J (1994) Experimental P NMR study of the influence of ionic strength on the apparent dissociation constant of MgATP. Magma 2:101-7. Murphy E, (2000) Mysteries of Magnesium Homeostasis. Circ Res 86:245-8.
2+

11

Human brain [Mg ]

2+

Chapter 1

Ramadan NM, Halvorson H, Vande-Linde A, Levine SR, Helpern JA, Welch KMA (1989) Low brain magnesium in migraine. Headache 29:590-3. Ramirez F, Marecek JF (1980) Coordination of magnesium with adenosine 5'-diphosphate and triphosphate. Biochim. Biophys Acta 589:21-9. Repke KRH (1982) On the mechanism of energy release, transfer, and utilization in sodium-potassium ATPase transport work: old ideas and new findings. Ann N Y Acad Sci 402:272-86. Skou C (1982) The sodium-potassium ATPase: coupling of the reaction with ATP to the reaction with sodium ion and potassium ion. Ann N Y Acad Sci 402:169-84.

Taylor JS, Vigneron DB, Murphy-Boesch J, Nelson SJ, Kessler HB, Coia L, Curran W., Brown TR (1991) Free magnesium levels in normal human brain and brain 31 tumors: P chemical-shift imaging measurements at 1.5 T. Proc Natl Acad Sci USA 88:6810-4. Welch KMA, Ramadan NM (1995) Mitochondria, magnesium and migraine. J Neurol Sci 134:9-14. Wells JA, Knoeber C, Sheldon MC, Werber MM, Yount RG (1980) Cross-linking of myosin subfragment 1. Nucleotide-enhanced modification by a variety of bifunctional reagents. J Biol Chem 255:11135-40. Williams GD, Smith MB (1995) Application of the accurate assessment of intracellular magnesium and 31 pH from the P shift of ATP to cerebral hypoxia- ischemia in neonatal rat. Magn Reson Med 33:853-7.

12

Magnesium in the Central Nervous System

Chapter 2

Intracellular magnesium homeostasis


Andrea M.P. Romani
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
amr5@po.cwru.edu

Abstract Magnesium (Mg2+) is the fourth most abundant cation in the whole body and the second most abundant cation within the cell. Numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signalling pathways are regulated by Mg2+. Our understanding of how cells regulate Mg2+ homeostasis and transport has registered significant progress in recent time. Yet, several aspects of Mg2+ homeostasis within cellular organelles, and the nature of the Mg2+ extrusion mechanisms at the cell membrane, are still undefined. The present work attempts to provide a comprehensive and updated review of the mechanisms regulating cellular Mg2+ homeostasis in eukaryotic cells under physiological conditions and the modifications these mechanisms undergo in various human and animal pathologies.

Introduction Mammalian cells contain high concentrations of total and free magnesium ion (Mg2+). These concentrations are essential to regulate numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signalling pathways. While the increasing number of observations supports a key regulatory role for Mg2+ within the cell, our understanding of how Mg2+ homeostasis is regulated at the cellular and subcellular level remains sketchy and incomplete. There are both conceptual and methodological reasons for this limitation. The relative slow turn- over of Mg2+ across the plasma membrane or other biological membranes in the absence of metabolic and hormonal stimuli, the absolute abundance of total and free Mg2+ within the cell, and the limited occurrence of significant changes in free [Mg2+] have all contributed for a long time to the assumption that cellular Mg2+ concentration does not change significantly, and is consistently at a level adequate for its role as a co-factor for various cellular enzymes and proteins. Consequently, this conceptual assumption has limited the interest to develop techniques and methodologies able to rapidly and accurately quantify changes in cellular Mg2+ content. In the last two decades, however, an increasing number of experimental and clinical observations have challenged this assumption. More than 1000 entries in the literature highlight the regulatory role of Mg2+ on various cellular functions and

cycles, and indicate the occurrence of major fluxes of Mg2+ across the plasma membrane of various mammalian cells under a variety of metabolic or hormonal stimuli. In turn, these fluxes have resulted in appreciable changes in cytosolic free [Mg2+] and total Mg2+ content within the cell and cellular organelles. Furthermore, genetic and electrophysiological approaches in bacteria, yeast and mammalian cells have identified several Mg2+ entry mechanisms that operate either at the cell membrane level or in the membrane of cellular organelles such as mitochondria and Golgi. At the same time, the increased interest in biological functions regulated by Mg2+ has stimulated the development of methodological approaches aimed at better detecting and quantifying variations in cellular Mg2+ level, and renewed interest in relating alterations in Mg2+ homeo- stasis with the onset of specific pathologies and complications in human patients. All these different aspects will be elucidated in the present review to provide a framework as comprehensive as possible to correlate changes in cellular Mg2+ homeostasis and content with variations in the function of specific enzymes, which ultimately affect the modus operandi of different cellular organelles and cell types. Cellular Mg2+ distribution Direct and indirect measurement of total cellular Mg2+ content by various techniques consistently 13

Intracellular Mg homeostasis

Chapter 2

indicates that total Mg2+ concentration ranges between 17 to 20mM in the majority of mammalian cell types examined (Romani and Scarpa, 1992; Wolf et al., 2003). Determinations of total and free Mg2+ concentrations by electron probe X-rays microanalysis (EPXMA), 31P-NMR, selective Mg2+-electrode, 13C-NMR citrate/iso- citrate ratio or fluorescent indicators (Table I in Romani and Scarpa, 1992, and in Wolf and Cittadini, 2003) localize major amounts of Mg2+ within mitochondria, nucleus, and endo-(sarco)- plasmic reticulum, with total concentrations ranging between 15 to 18 mM in each of these organelles. Binding of Mg2+ by phospholipids, proteins, nucleic acids, chromatin and nucleotides has been invoked to explain the persistence of such a high Mg2+ concentration within these organelles. Although the specific modality and nature of these bindings have not been fully investigated, experimental evidence indicates that only a small fraction of such a large Mg2+ content is actually free in the lumen of these structures. Concentrations of 0.8 and 1.2 mM free [Mg2+] have been measured in the matrix of cardiac and liver mitochondria (Jung et al., 1990; Rutter et al., 1990). No determinations of free [Mg2+] are available for the nucleus and the endo-(sarco)-plasmic reticulum. The porous structure of the nuclear envelope makes it reasonable to envision that the intranuclear free [Mg2+] is similar to the concentration measured in the cytoplasm. The free [Mg2+] within the endo- (sarco)-plasmic reticulum lumen cannot be reliably determined due to the elevated milli- molar concentration of Ca2+ inside the organelle (Somlyo et al., 1985), and the high affinity of the fluorescent dyes Mag-Fura or Mag-Indo for Ca2+ (50mM) as compared to Mg2+ (1.5 mM) (Hofer and Machen, 1993). A third considerable pool of Mg2+ (~4-5mM) is present in the cytoplasm in the form of a complex with ATP, and other phospho- nucleotides and phosphometabolites (Scarpa and Brinley, 1981). Because of its abundance (~ 5mM) and Mg2+ binding affinity (Kd ~78M), ATP constitutes the largest metabolic pool able to bind Mg2+ within the cytoplasm and the mito- chondria matrix as well (Luthi et al., 1999). The binding/buffering capacity of ATP, phospho- nucleotides and phosphometabolites, and possibly proteins, maintains cytosolic free [Mg2+] between 0.5-1mM, or less than 5% of total cellular Mg2+ content in almost all the cells and tissues examined (Table I in Romani and Scarpa, 14

1992). Similar values have been obtained using fluorescent dyes, 31P-NMR and citrate/isocitrate ratio (Romani and Scarpa, 1992). Overall, these results support the presence of a very limited chemical Mg2+ gradient across the cell membrane, and across the membrane of cellular organelles. In erythrocytes, which lack cellular Mg2+ compart- mentation, Flatman and Lew (1981) have observed three kinetically distinct binding pools for Mg2+: a low capacity, high affinity pool represented by cell proteins, and two pools that correspond reasonably well to ATP and 2,3- diphosphoglycerate (2,3-DPG) respectively (Gunther et al., 1995). This model has been further refined by Raftos et al., (1999) to take into account Mg2+ binding by hemoglobin under oxygenated and not oxygenated conditions. Limited information is available about the ability of proteins to bind Mg2+ within the cell and cellular organelles. Aside from calmodulin (Oki et al., 1997), troponin C (Wang et al., 1998), parvalbumin (Allouche et al., 1999), and S100 protein (Ogoma et al., 1992), there is no indication as to whether other cytosolic or intra- organelle proteins can bind substantial amounts of Mg2+ and contribute to the elevated concen- trations of total Mg2+ measured within the mitochondria or discrete regions of the endo- (sarco)-plasmic reticulum. An early report by Bogucka and Wojitczak (1971) has suggested the presence of two proteins able to bind Mg2+ with high affinity/low capacity and high capacity/low affinity, respectively, in the intermembraneous space of the mitochondrion. However, no subsequent study has confirmed their presence or identified the proteins. The presence of Mg2+ binding sites has been indicated for several other cellular proteins, but no clear information is available as to whether these proteins do bind Mg2+ under basal conditions and whether binding changes to a significant extent following hormonal or metabolic stimuli, or under pathol- ogical conditions. Moreover, the potential physiological relevance of Mg2+ binding by any of the mentioned proteins has been called into question by the observation that parvalbumin null mice do not exhibit hypomagnesaemia or significant changes in tissue Mg2+ handling and homeostasis (Belge et al., 2007). Taking into account the cellular distribution and assuming a Mg2+ concentration in plasma and

Magnesium in the Central Nervous System

Chapter 2

extracellular fluid of 1.2-1.4 mM, one-third of which is binding extracellular proteins (e.g. albumin) or other biochemical moieties (Geigy, 1984), it appears that most mammalian cells are at or near zero trans condition as far it concerns the chemical free [Mg2+] concentration across the cell membrane or the biomembrane of cellular organelles (e.g. mitochondria). Because the electrochemical equilibrium potential for cellular free [Mg2+] is approximately 50mM in most mammalian cells under resting conditions (Flatman, 1984), it is evident that mechanisms must operate in the cell membrane to maintain cytosolic free Mg2+ and total cellular Mg2+ content within the measured levels. Mg2+ transport mechanisms Eukaryotes retain their cellular Mg2+ content virtually unaltered under resting conditions even when exposed to a significant gradient across the cell membrane (e.g. culturing in virtually zero extracellular Mg2+ content) (Wolf et al., 2003; Romani, 2007). Atomic absorbance spectro- photometry determinations and radioisotopic equilibrium indicate Mg2+ turnover rates ranging from 1 hour (adipocytes) to several days (lymph- ocytes) as a result of structural and functional specificity of different tissues and cells (Romani, 2007). Within the same cell types discrepancies can be observed based upon the experimental conditions or the modality of isolation, e.g. cells in situ versus freshly isolated cells versus cells in culture. For example, cardiac ventricular myo- cytes attain 28Mg equilibrium within 3 hours in the whole animal but require 72-80 hours, as dispersed cells incubated at 37oC, or even a longer period of times when exposed to 20oC (Polimeni and Page, 1974; Rogers et al., 1960; Rogers, 1961). Lymphocytes also present differences in amplitude (or operation) of Mg2+ transport depending on whether they are freshly isolated (Wolf et al., 1997) or cultured (Maguire and Erdos, 1978) cells. For a long time, the slow Mg2+ turnover observed in various tissues or cells has contributed to the erroneous idea that Mg2+ content in mammalian cells does not change, or changes at such a slow pace that it lacks physiological significance. In the last twenty years, this notion has been completely reverted by a large body of experimental evidence, which indicates that large

fluxes of Mg2+ can cross the plasma membrane of eukaryotic cells within minutes from the application of metabolic or hormonal stimuli, with relatively small changes in free Mg2+ level (Romani, 2007; Grubbs and Maguire, 1987; Romani and Scarpa, 2000; Romani and Maguire, 2002). Lymphocytes (Gunther and Vormann, 1990; Wolf et al., 1997), erythrocytes (Matsuura, 1993), cardiac myocytes (Vormann and Gunther, 1987; Romani and Scarpa, 1990a) and liver cells (Jakob et al., 1989; Romani and Scarpa, 1990b; Gunther et al., 1991) are just a few examples of the mammalian cells that have been reported to extrude 10% to 20% of their total cellular Mg2+ content in less than 10min from the application of adrenergic or metabolic stimuli. The amplitude and rapidity of these fluxes presuppose the presence and operation of powerful transport mechanisms able to move large amounts of Mg2+ in and out of mammalian cells following various stimuli (for a list of experimental models and conditions see Romani and Scarpa, 2000). In addition, the operation of Mg2+ entry mechanisms appears to be tightly coupled with the ability of the cell to rapidly and efficiently buffering the magnesium ions accumulated, as suggested by the limited changes in cytosolic free [Mg2+]i in the large majority of conditions tested (Fatholahi et al., 2000; Kubota et al., 2005). The Mg2+ transport mechanisms operating at the level of the cell membrane or in the membrane of cellular organelles are represented by channels and exchangers. While channels are predominantly involved in Mg2+ accumulation, exchangers mediate essentially Mg2+ extrusion. The majority of recently identified Mg2+ entry mechanisms operate in the cell membrane but two of them have been located in the mitochondrial membrane and the Golgi system, respectively. For the most part, these entry mechanisms present a modest selectivity for Mg2+, and do not appear to be involved in Mg2+ extrusion. No information is currently available about the mechanisms that contribute to maintain an elevated Mg2+ concentration within the lumen of the endoplasmic (sarcoplasmic) reticulum. What it follows is a general description of our current knowledge about the channels and exchangers involved in Mg2+ transport across biological membranes (summarized in Table 1). 15

Intracellular Mg homeostasis

Chapter 2

Channels Magnesium ions enter the cell via channels or channel-like mechanisms. Channels able to transport Mg2+ into the cell have been originally described in prokaryotes (Kehres et al., 1998; Moncrief and Maguire, 1999), including proto- zoans (Preston, 1990), but recently several Mg2+ entry mechanisms with channels or channels-like features have been identified in eukaryotic cells. Some of these mechanisms exhibit a relatively high specificity for Mg2+ but they can permeate other divalent cations as well. Whereas the majority of these channels are located in the cell membrane or perhaps translocate between early endosomal vesicles and the cell membrane, other are specifically located in the mitochondrial membrane or in the Golgi cysternae. Because the identification and characterization of these eukaryotic Mg2+ transporting channels is far from being complete, information relative to their regulation is still largely fragmentary. The abundance of mechanisms favouring Mg2+ entry into the cell also raises the question as to what extent the different mechanisms cooperate to modulate Mg2+ entry or exert an absolute (or relative predominance) in specific cells under well defined conditions. TRPM Channels TRPM7 (Nadler et al., 2001) and TRPM6 (Schlingmann et al., 2002) were the first channels identified as being able to transport Mg2+ into mammalian cells. While Fleig and her group (Nadler et al., 2001) reported a preferential Mg2+ permeation through the LTRPC7 channel (i.e. TRPM7 based on the current nomenclature), genetic analysis (Schlingmann et al., 2002) indicated, more or less at the same time, that TRPM6, another member of the melastatin subfamily of TRP channels, exhibits a selective Mg2+ permeation. At variance of the ubiquitous nature of TRPM7, TRMP6 is specifically localized in the colon and the distal convolute tubule of the nephron, a distribution that strongly emphases the role of TRPM6 in controlling whole body Mg2+ homeostasis via intestinal absorption and renal resorption. In contrast, it would appear that TRPM7 is more in control of Mg2+ homeostasis in individual cells. The original observations have led to a flurry of studies aimed at better understanding the role, regulation and interaction of these channels with 16

other cellular components involved to a varying degree in Mg2+ homeostasis, and presently more than 190 publications relative to TRPM7 and 110 publications relative to TRPM6 can be found in the literature. Although sharing several similarities in terms of structure and operation, these two channels differ in various aspects ranging from location to hormonal modulation. TRPM7 The key role of TRPM7 in transporting Mg2+ into cells and modulating cell growth was first evidenced by Nadler et al., (2001). At the time, the channel was identified as LTRPC7 or long TRP channel 7 owing to the presence of a particularly long extension outside the channel segment (Yamaguchi et al., 2001). Due to the presence of an alpha-kinase domain at the C-terminus (Ryazanova et al., 2001) and its functional homology to eEF2-kinase (Ryazanov, 2002), this protein was already known as CHAK1 (channel kinase 1) (Ryazanova et al., 2001). Shortly after the observation of Nadler et al., (2001), Runnels et al., (2001) evidenced the peculiar structure of TRPM7, combining a channel structure with an alpha-kinase domain at the C-terminus. Although originally investigated for a possible role in Ca2+ signalling in lymphocyte, it became rapidly apparent that the channel would preferentially carry Mg2+ and Ca2+ (Nadler et al., 2001) but also trace amounts of divalent cations such as Ni2+ and Zn2+ (Bessac and Fleig, 2007; Monteilh-Zoller et al., 2003). Located on the human chromosome 15 at the locus 15q21, the protein is formed by 1865 amino acids arranged to possibly form 10 trans- membrane domains, with both the C- and N- termini internalized. The protein is ubiquitously expressed albeit to a varying extent in all mammalian cells tested so far. The functional structure is supposed to be a tetramer but disagreement exists as to whether it is formed by 4 identical monomers or by a combination of TRPM7 and TRPM6 (see following section) arranged with a varying stoichiometry in different portion of the cell membrane or in different cell types. Voets and colleagues reported the functional expression of TRPM6 channels in HEK- 293 cells with electrophysiological properties similar to those of TRPM7 (Voets et al., 2004). In contrast, Chubanov et al., (2004) reported the absence of functional currents through TRPM6

Table 1. Mg2+ transporters in eukaryotes Family


Members TRPM6 TRPM7 Claudin 16 (PCLN-1) Claudin-19 MagT1

Apparent Km ~0.7 mM ~0.7 mM ~0.7mM ~0.7mM 0.2 mM

Type of Transporter Channel Channel Channel Channel Channel

Entry Mechanisms

TRPM

Reference Schlingmann et al., 2002 Nadler et al., 2001 Simon et al., 1999 Hou et al., 2009 Goytain and Quamme, 2005a Zou and Clapham, 2009 Goytain and Quamme, 2005b Goytain and Quamme, 2005c Sahni et al., 2007 Goytain and Quamme, 2005d Goytain and Quamme, 2005d Goytain et al., 2007 Goytain et al., 2008a Goytain et al., 2008b Goytain et al., 2008b Koliske et al., 2003 Goytain and Quamme, 2008 Goytain and Quamme, 2008

Cell Membrane

Claudins

MagT1

SLC41

SLC41A1 SLC41A2

0.7 - 3 mM 0.7 - 3 mM

Carrier Carrier

ACDP NIPA Huntingtin Mrs2 MMgt

ACDP1 ACDP2 NIPA1 (SPG6) NIPA2 Huntingtin1 (HIP14) HIP14L Mrs2/AtMrs2, Lpe10 MMgT1 MMgT2

~0.7 mM ~0.5 mM 0.7 mM 0.7 mM 0.87 mM 0.74 mM ~1.5 mM 1.5 mM 0.6 mM

Carrier Carrier Carrier Carrier Carrier Carrier Channel Channel Channel

Mitochondria Golgi

17

Family Exit Mechanisms


Members ND

Apparent Km 15-20 mM

Type of Transporter Antiport

Na+/Mg2+ exchanger

Reference Gunther and Vormann, 1984 Tashiro and Konishi, 1997 Cefaratti et al., 1998 Ebel et al., 2002 Kolisek et al., 2008 Shaul et al., 1999

Cell Membrane

Na+- independent SLC41 H+/Mg2+ exchanger

ND (choline?) SLC41A1 AtMHX#

~20mM ~0.7 mM ~15mM

Exchanger (?) Carrier Exchanger

# Identified only in plants and yeast and not in mammalian/human cells.

18

Magnesium in the Central Nervous System

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when the channel is expressed by itself in either HEK-293 cells or X. Laevis oocytes, and suggested that TRPM7 co-expression was required for TRPM6 to incorporate into channel complexes at the plasma membrane level. Schmitz et al., (2005) subsequently confirmed the association of TRPM6 and TRPM7 channel proteins to obtain a functional structure. Yet, a detailed functional characterization of the TRPM6/7 chimeric channel remained undefined (Chubanov et al., 2005) until Yue and collaborators addressed the issue in two elegant electro-physiology studies (Li et al., 2006; Li et al., 2007). In these studies, the authors demonstrate that TRPM6 and TRPM7 can indeed form a chimeric heterotetramer, and that TRPM6, TRPM7, and TRPM6/7 constitute three distinct ion channels with different divalent cation permeability, pH sensitivity, and unique single channel conductance. In addition, these authors reported that 2-APB can differentially regulate the channel activities of TRPM6, TRPM7, and TRPM6/7, markedly increasing Mg2+ and Ca2+ entry through TRPM6 (Li et al., 2006). Based on these results, it would then appear that TRPM6 can form either functional homotetrameric channels or hetero-tetrameric TRPM6/7 channels (Gwanyanya et al., 2004). A corollary of this observation would be that TRPM6, TRPM7, and TRPM6/7 channels may play different roles under various physiological or pathological conditions in different tissues. A detailed mapping of the distribution of homomeric TRPM7 versus heteromeric TRPM6/7 channels in various tissues, however, is still lacking, leaving their relative role largely undefined. More recently, some light has been shed on the modality of TRPM7 regulation. At the direct channel level, TRPM7 inward current is markedly enhanced by protons, which compete with Ca2+ and Mg2+ for binding sites, most likely at the level of the channel pore, thereby releasing the blockade of divalent cations on inward mono- valent currents (Bessac and Fleig, 2007; Monteilh- Zoller et al., 2003). Not only extracellular protons significantly increased monovalent cation permeability, but higher proton concentrations are required to induce 50% of maximal increase in TRPM7 currents under conditions in which extracellular Ca2+ and Mg2+ concentrations are increased. Following the increase in extracellular H+ concentration, in fact, the apparent affinity for Ca2+ and Mg2+ is significantly diminished. This set

of observation suggests that at physiological pH, Ca2+ and Mg2+ bind to TRPM7 and inhibit the monovalent cation currents. At higher H+ concentrations, instead, the affinity of TRPM7 for Ca2+ and Mg2+ is decreased, thus allowing monovalent cations to permeate the channel (Jiang et al., 2005). Another level of regulation appears to be provided by PIP2, as initially reported by Clapham and his collaborators (Runnels et al., 2002). This observation was not confirmed by Fleigs group, which instead reported a regulatory role by cAMP (Takezawa et al., 2004). More recently, however, Langeslag et al., (2007) have observed that the depletion in PIP2 level resulting from PLC-activation counteracts TRPM7 activation. It would therefore appear that either PLC-activation accelerates TRPM7 rundown via PIP2 depletion or PIP2 depletion plays a feedback regulatory role on the channel activation by PLC (Langeslag et al., 2007). Additional evidence for a regulatory role by PIP2 on TRPM7 has been provided by Mubagwas group (Gwanyanya et al., 2006; Macianskiene et al., 2008). This group, in fact, has reported that inhibition of phospholipase C or addition of exogenous PIP2 decreases the run-down of the channel whereas the extracellular addition of phenylephrine accelerates it (Macianskiene et al., 2008). In addition, this group has observed that both ATP (Gwanyanya et al., 2006) and non- hydrolysable GTP analogs modulate the channel activity, most likely by forming MgATP and by accelerating the channel run-down via phospholipase-C activation, respectively (Macianskiene et al., 2008). The regulatory role of PIP2 on TRPM7 is further emphasized by the experimental evidence that agonists like bradykinin or angiotensin-II, which activate phospholipase-C coupled receptors via Gq signalling (Touyz et al., 2006; Langeslag et al., 2007) can modulate the channel activity via PIP2 metabolism. Interestingly, TRPM7 activation only takes place in the presence of a physiological cellular [Mg2+]i, whereas reducing this concen- tration below its physiological level with EDTA- AM results in a PLC-mediated inactivation of TRPM7 activity, most likely via PIP2 depletion (Langeslag et al., 2007). The interaction between TRPM7 and phosphatidyl-inositol metabolites is further supported by the observation that TRPM7 is required for a sustained phosphoinositide-3- 19

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kinase signalling in lymphocytes (Sahni and Scharenberg, 2008). In the presence of a physiological concentration of extracellular Mg2+, TRPM7-deficient cells rapidly down-regulate their rate of growth as a result of a signalling deactivation downstream PI3-Kinase (Sahni and Scharenberg, 2008), the phenotype being rescued by supplementing the culture medium with Mg2+ (Sahni and Scharenberg, 2008). A structural peculiarity of TRPM7 is the presence of an alpha-kinase at the C-terminus, which specifically phosphorylates serine and threonines located in an alpha-helix (Middelbeek et al., 2010). Initial experimental evidence (Runnels et al., 2002) invoked an essential role of this kinase domain in modulating channel activation and gating. Subsequent studies, however, failed to fully support this initial claim, as they indicated that TRPM7 channels lacking the kinase domain could still be activated by internal Mg2+ depletion (Schmitz et al., 2003). One consequence of lacking the kinase domain, however, is the inability of properly phosphorylating and consequently activating downstream cellular components. Yet, little is know about the molecular mechanisms activating the kinase domain. A recent study by Clark et al., (2006) strongly suggests that autophosphorylation plays a significant role in target recognition by the TRMP7 kinase domain. Phosphomapping by mass spectrometry has confirmed the massing autophosphorylation of TRPM7 kinase domain, which in turn increases the rate of substrate phosphorylation. The phosphomapping has also identified the majority (37 out of 46) of the autophosphor-ylation sites in a Ser/Thr rich region immediately preceding the kinase catalytic domain (Clark et al., 2008). Deletion of this region does not affect the intrinsic catalytic activity of the kinase but prevents substrate phosphorylation, confirming the role of this region in substrate recognition (Clark et al., 2008). Although this Ser/Thr region is poorly conserved at the amino acid sequence in TRPM6, the kinase domain of this channel appears to require a similar massive autophos-phorylation of its Sr/Thr residues for proper substrate recognition and efficient target phosphorylation (Clark et al., 2008). So far, only annexin I (Dorovkov and Ryazanov, 2004), myosin IIA heavy chain (Clark et al., 2008a; 20

Clark et al., 2008b), and calpain (Su et al., 2006) have been clearly identified as substrates phosphorylated by TRPM7 kinase domain. Although the number of targets is rather restricted, it appears that TRPM7 is playing a double role within cells by regulating Mg2+ homeostasis on the one hand, and cellular functions centered on cell adhesion, contractility and (anti)-inflammatory processes on the other hand. This double role of TRPM7 within cells, in particular smooth muscle cells, is emphasized by a recent observation by Touyz and colleagues (Paravicini et al., 2009). In this study, the authors report that aortic segments of mice exhibiting low intracellular Mg2+ levels present increased medial cross-section and increased TRPM7 expression but decreased levels of annexin-I expression. As annexin-I has a major anti- inflammatory role (Parente and Solito, 2004), the results of this study suggest a potential regulatory role of TRPM7 in regulating vascular structure and integrity, as well as inflammation. As our understanding of TRPM7 expression and regulation has improved, evidence of a major functional role of the channel in neuronal function and survival under hypoxia or ischemic- reperfusion conditions has increased. Owing to its ability to transport either Ca2+ or Mg2+, TRPM7 exhibits an ambivalent role based upon the permeating cation. Following activation by reactive oxygen/nitrogen species and prolonged oxygen and glucose deprivation, TRPM7 favours Ca2+ fluxes that result in a toxic event for neurons (Aarts et al., 2003). In contrast, Mg2+ permeation of the channel enhances anti-apoptotic and cell- survival mechanisms, preventing the anoxic death of neurons (Clark et al., 2006). The essential role of TRPM7 in detecting extracellular divalent cations is supported by a recent study by Wei et al., (2007), which indicates that activation of the channel by low extracellular divalent cations is lethal to the cell. At the same time, Jiang et al., (2008) have reported that occlusion of the middle cerebral artery for 1 hour enhances TRPM7 expression in ipsilateral hippocampus, with deleterious consequences for the neurons. The increased expression of TRPM7 and its consequences are largely counteracted by pre- treatment with nerve growth factor via activation of TrkA pathway (Jiang et al., 2008). More recently, application of 5-lipoxygenase inhibitors can block TRPM7 current without affecting

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protein expression and cell membrane concentration, de facto preventing cell death (Chen et al., 2010). The involvement of TRPM7 is not restricted to the sympathetic nervous system but extends to the parasympathetic system as well, in which the channel facilitates the fusion of cholinergic vesicles with the plasma membrane without affecting large dense core vesicle secretion (Brauchi et al., 2008). Despite the data accumulated since its identification as a preferential Mg2+ channel, a recent report by Claphams group has cast some concern about the effective role of TRPM7 in regulating Mg2+ homeostasis (Jin et al., 2008). In their report, the authors indicate that TRPM7 null mouse present an altered embryonic develop- ment, and that tissue specific deletion of the channel in T cell lineage disrupts thymopoiesis, leading to the progressive depletion of thymic medullary cells. Deletion of TRPM7, however, did not affect acute accumulation of Mg2+ nor impacted total cellular Mg2+ content in T cells. The synthesis of several growth factors, however, was significantly dysregulated, resulting in an altered differentiation of thymic epithelial cells (Jin et al., 2008). TRPM7, therefore, appears to be the first TRP channel with a non-redundant and actually essential role in embryogenesis and thymopoiesis. Whether these defects are the result of an altered Ca2+ rather than Mg2+ homeostasis is presently undefined. It is also unclear how the removal of this protein results in an altered cellular differentiation process. TRPM6 The unique localization of TRPM6 channels in the colon and the renal distal convolute tubule, two epithelia otherwise highly impermeable to salt re- absorption, highlights the specific role of this channel in controlling intestinal Mg2+ absorption and renal Mg2+ resorption, and consequently contributing to whole-body Mg2+ homeostasis. The TRPM6 gene was originally identified as the site of various mutations responsible for Hypomagnesaemia with Secondary Hypocal- caemia (HSH), a rare autosomal recessive disease characterized by Mg2+ and Ca2+ wasting, whose symptoms could be ameliorated by massive intra-

venous Mg2+ administration followed by oral Mg2+ supplementation (Schlingmann et al., 2002). Surprisingly, while hypocalcaemia is completely alleviated by this treatment, the patients continue to present serum Mg2+ level around 0.5- 0.6 mmol/L, i.e. about half the physiological level (Schlingmann et al., 2002). Because the primary defect in these patients is at the level of the TRPM6 expressed in the intestine (Schlingmann et al., 2002), the excess Mg2+ supplementation is rapidly filtered at the glomerular level and increases passive renal absorption via paracellin- 1 (see next section). Trans-cellular absorption via renal TRPM6, however, remains depressed and unable to restore physiological serum Mg2+ level (Schlingmann et al., 2002). At the functional level, experimental evidence suggests that the channel forms a tetramer within the plasma membrane. As indicated in the previous section, questions remain as to whether the channel forms a homo-tetramer, or a hetero- tetramer with TRPM7, with a varying stoich- iometry. Irrespective of the possibilities, several TRPM6 mutations have been identified (Walder et al., 2002). The majority of these mutations result in the expression of a truncated and non- functional channel (Walder et al., 2002). The missense mutation S141L, on the other hand, occurs at the N-terminus of the channel and prevents its proper assembly as a homo- tetramer, or a hetero-tetramer with TRPM7 (Walder et al., 2002). Another missense identified in humans is the P1017R mutation (Walder et al., 2002), which appears to occur in a region putatively identified as the pore region of the channel. Yet, this mutation affects negatively and more significantly TRPM7 function when this protein is co-expressed with TRPM6 (Walder et al., 2002). More recently, TRPM6 null mice have been developed by Sheffield and his collaborators (Walder et al., 2009). The heterozygous Trpm6+/- have for the most part normal electrolyte levels aside for a modest low plasma Mg2+ level (~0.67 vs. 0.75, (Walder et al., 2009). The majority of the homozygous Trpm6-/- animals die by embryonic day 12.5. Most of the few animals that survive to term present significant neural tube defects, consisting primarily of both exencephaly and spina bifida occulta. Administration of high Mg diet to dams improves offspring survival to weaning (Walder et al., 2009). 21

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A peculiarity TRPM6 shares with TRPM7 is the presence of an alpha-kinase domain at the C- terminus. Originally, TRPM6 was known as CHAK2 (channel kinase 2) (Ryazanov, 2002) due to the presence of this kinase domain, which presents a functional homology to eEF2-kinase (Ryazanov, 2002). At variance of other kinases, this domain phosphorylates serine and threonine residues located within an alpha-helix instead of a beta- sheet (Ryazanova et al., 2001; Ryazanov, 2002; Middelbeek et al., 2010). Owing to their dual function as a channel and a kinase, TRPM6 and TRPM7 are currently referred to as chanzymes. As in the case of TRPM7, removal of the kinase domain does not abolish entirely the channel activity but modulates the extent to which the channel is regulated by intra-cellular free Mg2+ or MgATP complex (Chubanov et al., 2004; Schmitz et al., 2005; Chubanov et al., 2005; Li et al., 2005; Thebault et al., 2008). Hence, the targets phosphorylated by the kinase must be located downstream from the protein. At variance of what reported for the kinase domain of TRPM7 (see previous section) no phosphorylation substrate for the TRPM6 kinase has been clearly identified up-to-date, with the exception of TRPM7 itself. Data from the Ryazanovs group clearly indicate that the TRPM6 kinase domain can phosphorylate TRPM7 channel within a heterotetramer structure while the opposite does not occur (Schmitz et al., 2005). Due to the limited information available, it is largely undefined as to whether the kinase domain of TRPM7 and TRMP6 phosphorylate similar or different substrates in the tissues in which these chanzymes are specifically expressed. How exactly the expression and/or activity of TRPM6 channel are modulated in vivo is slowly being elucidated. Estrogens (17-estradiol) markedly upregulate TRPM6 mRNA in both colon and kidney while having no effect on TRPM7 mRNA (Groenestege et al., 2006; Cao et al., 2009). In the absence of estrogen, the repressor of estrogen receptor activity (REA) binds to the 6th, 7th and 8th beta-sheets of TRPM6 kinase domain in a phosphorylation-dependent manner and inhibits TRPM6 activity (Cao et al., 2009). Short-term estrogen administration dissociates the binding between REA and TRPM6, resulting in an increased channel activity (Cao et al., 2009). Dietary Mg2+ restriction also upregulates TRPM6 mRNA in both colon and kidney but does not 22

affect TRPM7 mRNA (Groenestege et al., 2006; Rondon et al., 2008). In contrast, an Mg2+ enriched diet upregulates TRPM6 mRNA only in the colon, in keeping with an increased intestinal absorption (Groenestege et al., 2006). Mice selected for the their low erythrocyte and plasma Mg2+ status exhibit hypomagnesaemia and hypomagnesuria, and increased TRPM6 expression in kidney and intestine when fed a severely Mg2+-deficient diet (Rondon et al., 2008). Feeding the mice an Mg2+ adequate diet resulted, instead, in hypomagnesaemia and hypermag- nesuria, and lower intestinal and renal TRPM6 expression (Rondon et al., 2008). These changes in TRPM6 expression and Mg2+ level in blood and urine were not observed in mice exhibiting normal or high erythrocyte and plasma Mg2+ level (Rondon et al., 2008). It is becoming progressively apparent, therefore, that genetic factors control TRPM6 expression and activity, and that dietary Mg2+ restriction increases Mg2+ resorption, the process correlating well with an increased TRPM6 expression in both intestine and kidney (Groenestege et al., 2006; Rondon et al., 2008). As already observed for TRPM7, intracellular ATP specifically decreased TRPM6 current (Chubanov et al., 2004; Schmitz et al., 2005; Chubanov et al., 2005; Li et al., 2005; Thebault et al., 2008). The inhibitory site resides in the conserved ATP- binding motif GXG(A)XXG within the alpha-kinase domain (Thebault et al., 2008). Either the full deletion of the kinase domain or point mutations within the ATP-binding motif (G1955D) completely prevents the inhibitory effect of intracellular ATP. The effect of ATP, however, is independent of alpha-kinase autophosphorylation activity (Thebault et al., 2008). The activity of TRPM6 channels can also be modulated by cellular signalling molecules. Bindels and collaborators (Cao et al., 2008) have reported that over-expression of RACK1 (receptor for activated protein kinase C) results in a direct binding of this protein to the alpha-kinase domain of TRPM6, and possibly TRPM7 due to the high homology (>84%) between the two kinase domains. The TRPM6 site binding RACK1 is restricted to the region between amino acids 1857 and 1885 (6th, 7th and 8th b sheets). Interestingly, these are the same sheets involved in REA regulation (Cao et al., 2009). The interplay between REA and RACK1 in modulating the

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channel activity, however, still remains undefined. Accessibility analysis of the RACK1 binding site suggests that 18 of the 28 amino acids of this site are localized at the surface of the TRPM6 alpha-kinase domain (Cao et al., 2008). As a result of this interaction, the channel activity of TRPM6 and TRPM7 are inhibited. As it could be anticipated, co-expression of RACK1with a alpha- kinase deleted TRPM6 mutant fails to suppress channel activity. The inhibitory effect of RACK1 fully depends on the autophosphorylation of threonine 1851 (T1851) within the kinase domain. This residue is localized at the end of the 4th alpha-helix adjacent to the RACK1 binding site. Mutation of T1851 to alanine (T1851A) or to aspartate (T1851D) significantly decreases TRPM6 autophosphorylation while leaving unaltered RACK1 binding. The inhibitory effect of RACK1 on the channel activity, however, is completely abolished only in the case of T1851A mutation while it persists in the case of T1851D mutation (Cao et al., 2008). Interestingly, T1851D autophosphorylation strongly depends on Mg2+ concentration, steadily increasing for concentrations between 0.1 to 1mM. In contrast, T1851A mutant is less sensitive to intracellular Mg2+ concentrations as compared to the wild type (IC50 ~0.7 vs. 0.5mM, respectively). Under conditions in which protein kinase C is activated (e.g. pre-incubation with PMA), the inhibitory effect of RACK1 on TRPM6 channel activity is completely prevented (Cao et al., 2008). The inhibition, however, can be restored by pre- treatment with the PKC inhibitor chelerythrine (Cao et al., 2008), suggesting a competing effect of PKC for RACK1. A recent study of Groenestege et al., (2007) has evidenced the role of EGF as an autocrine/ paracrine magnesiotropic hormone. By engaging its receptor in the basolateral domain of the distal convolute tubule, EGF is able to activate TRPM6 at the apical domain of the cell and induce cellular Mg2+ accumulation. Point mutation in the pro-EGF (P1070L) retains EGF secretion to the apical membrane and disrupts this cascade of events, ultimately resulting in the Mg2+ wasting typical of isolated recessive renal hypo-magnesemia (IRH). An alteration of the axis EGF/TRPM6/Mg2+ reabsorption/renal Mg2+ wasting is also observed in cancer patients undergoing treatment with antibodies anti-EGFR (Cunningham et al., 2004; Dimke et al., 2010), as

the antibody antagonizes the stimulation of TRPM6 activity via EGF. The modality by which EGF modulates TRPM6 activity and/or expression appears to involve ERK1/2 signalling. A report by Ikari et al., (2008) indicates that the stimulation of NRK-52E cells by EGF results in an increased phosphorylation of ERK1/2 and an increased expression of TRPM6 in a time-dependent manner (Ikari et al., 2008) via modulation by adaptin protein-1 (AP-1) (Ikari et al., 2010). The process is prevented by the use of antagonists for integrin avb3 or for MEK1/MEK2 activity, or by the use of siRNA for TRPM6 (Ikari et al., 2008). How exactly EGF, integrin, and ERK1/2 interact to enhance TRPM6 expression needs further elucidation. It is in fact unclear whether the activation of this signalling axis is connected to the release of RACK1-mediated inhibition of TRPM6 activity mentioned previously (Cao et al., 2008). The modality by which apically accumulated Mg2+ is transported across the cell to be delivered to basolateral domain of the cell to be extruded into the blood stream also needs further elucidation. One hypothesis is that parvalbumin and calbindin-D28k, two proteins abundantly present within cells of the distal convolute tubule of the nephron, can operate the transcellular transport of Mg2+ accumulated at the apical domain, or at least accelerate the rate of delivery at the basolateral domain. However, as mentioned previously, parvalbumin null mice do not show detectable defects in Mg2+ excretion or homeostasis (Belge et al., 2007), de facto questioning whether parvalbumin does play a role in the process or other proteins can compensate for its absence in the null model. Claudins Paracellin 1 (claudin 16) was the first Mg2+ transporting protein to be identified in mammals (Simon et al., 1999). The identification was rendered possible by the genetic analysis of patients affected by Familial Hypomagnesaemia with Hypercalciuria and Nephrocalcinosis (FHHNC), a disease characterized by massive renal Mg2+ and Ca2+ wasting that leads rapidly and irreversibly to renal failure (Simon et al., 1999). At variance of what is described for patients with TRPM6 mutations, the symptoms and the progressive renal deterioration in FHHNC patients are not ameliorated by Mg2+ supplementation (Simon et al., 1999). The gene responsible for this disease was identified by 23

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Lifton and collaborators in 1999 and named Paracellin-1 (PCLN-1) (Simon et al., 1999). More than 20 mutations affecting paracellin-1 trafficking or permeability have been identified up-to-date (Kausalya et al., 2006). PCLN-1 encodes for paracellin-1 (PCLN-1), also termed claudin-16. This protein is a member of the claudin family ((Lal-Nag and Morin, 2009), which comprehends a group of tight junction proteins that present 4 transmembrane spans coordinated by 2 extracellular loops, and both C- and N- termini on the cytoplasm side. Claudin-16 mediates paracellular Ca2+ and Mg2+ fluxes throughout the nephron. Yet, discrepancy exists about the modality by which these fluxes are generated. Data obtained in LLC-PK1 (a porcine renal cell line) suggest that claudin-16 mediates paracellular Na+-permeation which, in turn, generates a positive potential within the lumen that acts as the driving force for Mg2+ and Ca2+ resorption (Hou et al., 2005). In contrast, data in MDCK cells indicate a decrease in Na+- permeability and an increase in Mg2+ permeability (Ikari et al., 2006). It is unclear whether these discrepancies reflect a different modus operandi in cell lines of differing origin, or depend on the experimental conditions utilized in the two studies. It is evident, however, that PCLN-1 expression is modulated based upon the magnesium concentration present in the extracellular medium (Efrati et al., 2005). At the functional level, claudin-16 has to be delivered correctly to the tight junction where it interacts with the scaffolding protein ZO-1 (Muller et al., 2003). The association and dissociation of claudin-16 and ZO-1 appear to be regulated via PKA-mediated phosphorylation of Ser217 in claudin-16 (Muller et al., 2003). The dephosphorylation of this residue, as it occurs upon activation of the Calcium Sensing Receptor (CaSR) (Khan and Conigrave, 2010) results in the dissociation of claudin-16 from ZO-1 and its accumulation in the lysosomal compartment (Ikari et al., 2006). Mutations of Ser 217 can therefore accelerate claudin-16 turnover and modulate its function. Mutation of Threo233 (T233R) also impairs the interaction between claudin-16 and ZO-1, and favours the accumulation of claudin-166 into lysosomes (Muller et al., 2003; Ikari et al., 2006). More recently, evidence has emerged indicating the 24

involvement of another claudin isoform, claudin- 19, in mediating Mg2+ and Ca2+ resorption (Hou et al., 2009) by forming a head-to-head cation- selective complex with claudin-16 at the level of the tight junction. While the channel function of claudin-16 may not depend on its association with claudin-19, claudin-19 plays an indispensable role in recruiting claudin-16 to form a co-polymer at the level of the tight junction and in switching the channel from anion to cation selective (Hou et al., 2009). The heteromeric association between claudin-16 and claudin-19 is dramatically affected by point mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) and claudin-19 (L90P and G123R), which abolish the physiological synergism between the two proteins and result in the development of FHHNC. MagT1 This protein was identified by Goytain and Quamme (2005a) in human epithelial cells that upregulate the encoding gene following exposure to low-Mg2+ concentrations in the culture medium. The protein encoded by this gene has an estimated molecular weight of 38 KDa and 5 transmembrane domains. The mature MagT1 protein, however, would contain 4 transmembrane spans owing to the cleavage of the first transmembrane segment located near the C-terminus. At variance of SLC41 (Section 3.3.1) and Mrs2 (discussed in the next Section), MagT1 does not exhibit any significant degree of homology to prokaryotic Mg2+ transporters, but has some similarities with the oligosaccharide transferase complex OST3/OST6 that regulates protein glycosylation in the endoplasmic reticulum in yeast (Shibatani et al., 2005). The murine orthologue of MagT1 is highly expressed in liver, heart, kidney and colon, with detectable levels in lung, brain and spleen (Goytain and Quamme, 2005a). For the most part, MagT1 levels in these tissues are consistent with the mRNA levels, the only exception being the liver in which a low protein level is detected (Goytain and Quamme, 2005a). At variance of the other transporters described in this section, MagT1 appears to possess high specificity for Mg2+ (Km = 0.23mM). The Mg2+-elicited currents are not inhibited by Ca2+ but can be inhibited by Ni2+, Zn2+ and Mn2+, although the required concentrations (>0.2mM) far exceed the physiological concentrations of these cations in extracellular

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fluids. Interestingly, nitrendipine at ~10mM can inhibit the Mg2+ current whereas the more common nifedipine does not, even at much higher concentrations (Goytain and Quamme, 2005a). Virtually no information is available about N33, a second member of the MagT family. Although able to transport Mg2+, this protein does not show the same high specificity presented by MagT1 for the cation, and can also mediate the transport of Fe2+, Mn2+ and Cu2+ (Goytain and Quamme, 2005a). Based upon these observations, MagT1 appears to possess channel-like characteristics and a high selectivity for Mg2+. The latter evidence strongly suggests that this transporter can play an essential role in regulating Mg2+ homeostasis in mammalian cells. Support to this hypothesis is provided by the report of Zhou and Clapham (2009) that knock-out of MagT1, and its human homolog TUSC3, in HEK-293 cells results in a major reduction of cellular Mg2+ content. These authors also provide evidence that either protein can complement the yeast Mg2+ transporter ALR1 (Zhou and Clapham, 2009). Interestingly, the mRNA levels of MagT1 but not those of TUSC3 increase markedly following exposure of expressing cells to low extracellular Mg2+ concentrations for 1 day and 2 days. Incubation of the cells in high extracellular Mg2+ concentration has no effect on the expression of either protein (Zhou and Clapham, 2009). Mrs2 This protein was identified during a screening aimed at isolating nuclear genes suppressing RNA splicing defects in yeast mitochondrial introns (Wiesenberger et al., 1992). The three main characteristics observed in yeasts deficient in Mrs2 are: 1) a splicing phenotype, 2) a significant reduction in cytochromes, and 3) a deficit in mitochondria respiration to the point that the yeasts become unable to grow on non- fermentable substrates. Structurally, Mrs2 shows short regions of homology to the bacterial transporter CorA (Bui et al., 1999), and shares a similar membrane topology with 2 transmembrane domains. Mutants lacking Mrs2 can be rescued by CorA fused to the mitochondrial N-terminus leader sequence of Mrs2, which guarantees proper insertion in the mitochondrial membrane. These

mutants also present a decrease in mitochondrial Mg2+ content, which strongly supports a key role of Mrs2 in regulating mitochondrial Mg2+ homeostasis. Studies carried out with the fluorescent indicator Mag-Fura indicate a marked decrease in mitochondrial matrix Mg2+ level in yeast lacking Mrs2 protein whereas the over- expression of the protein results in a rapid and marked increase in matrix free Mg2+ (Kolisek et al., 2003). Mrs2 apparently functions as a channel, and its function is modulated by mitochondrial Dy as well as by inhibitors of F0-F1- ATPase or ANT, which substantially decrease Mg2+ influx. Highly conserved motifs in the middle region of the protein, corresponding to the coiled-coil portion of the channel, appear to be essential to form functional channels, or to gate the channel. More recently, Schweyen and his collaborators (Piskacek et al., 2009) have confirmed in HEK293 cells some of the mitochondrial modifications observed in yeasts. These authors, in fact, have reported that HEK 293 cells deprived of Mrs2 (Piskacek et al., 2009) lack complex I expression in mitochondria and present reduced level of mitochondrial Mg2+. Furthermore, the cells show a change in configuration as well as an increased incidence in apoptosis, which within 2 weeks results in a complete loss of cell viability (Piskacek et al., 2009). It still remains to be elucidated whether the decrease in mitochondrial Mg2+ simply depends on the absence of Mrs2, or is related to some extent to the absence of complex I, which affects mitochondrial Dy and consequently Mg2+ retention within the organelle (Akerman, 1981). Mammalian cells express a single orthologue of Mrs2, which can rescue Mg2+ deficient yeast strain (Zsurka et al., 2001). Hence, it appears that the mammalian/human Mrs2 homologue functions in a manner similar to the yeast homologue in mediating Mg2+ entry in mitochondria. Under conditions in which Mrs2p is absent, the operation of an alternative but much slower mitochondrial Mg2+ entry mechanism is observed. Although this pathway restores Mg2+ homeostasis only partially, it does rescue the phenotype of Mrs2 deficient yeast, ensuring their survival. No information is presently available about the identity, abundance, and regulation of this alternative transporter in mitochondria. Taken together, the data on Mrs2 suggest that Mg2+ is dynamically regulated within the 25

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mitochondrion, in which it plays a significant role in modulating mitochondrial dehydrogenases and oxygen consumption (Panov and Scarpa, 1996a; Panov and Scarpa, 1996b). MMgTs This gene family comprehends two proteins termed MMgT1 and MMgT2 (for membrane Mg2+ transporter 1 and 2) by Goytain and Quamme, who identified them by microarray analysis screening (Goytain and Quamme, 2008). The chromosomal location of these proteins in the mouse is XA5 for MMgT1 and 11B2 for MMgT2. In the rat, the respective locations are Xq36 for MMgT1 and 10q23 for MMgT2. Human MMgT1 is instead located on Xq26.3 (Goytain and Quamme, 2008). Immunohistochemistry assessment indicates that MMgT1 and MMgT2 are essentially located in the Golgi complex and post-Golgi vesicles, where they may contribute to the regulation of Mg2+ dependent enzymes involved in protein assembly and glycosylation (Goytain and Quamme, 2008). This localization, however, does not exclude that these proteins may play a role in modulating Mg2+ homeostasis at sites downstream the Golgi network. Widely distributed in tissues, these proteins are formed by 131 (MMgT1) and 123 (MMgT2) amino acids assembled into two predicted transmembrane domains. This suggests that these proteins can form homo-oligomeric and possibly hetero- oligomeric channels to favour Mg2+ permeation. MMgT-mediated Mg2+ uptake is saturable with a Km ~1.5mM for MMgT1 and ~0.6mM for MMgT2, and these values do not vary significantly with voltage. However, MMgT1 and MMgT2 are not specific for Mg2+ but they can transport other cations as well. Some slight differences in cation permeation exist between the two isoforms. Whereas MMgT1 mediates Sr2+, Fe2+, Co2+ and Cu2+ transport in addition to Mg2+, MMgT2 favours Sr2+, Co2+, Cu2+, Ba2+ and Mn2+ transport (Goytain and Quamme, 2008). Electrophysiological experiments indicate that Mg2+-generated current in MMgT1 are inhibited by 0.2mM Mn2+ but not by Gd3+ or Ni2+. Consistent with what reported for other Mg2+ transport mechanisms, MMgT1 mRNA increases ~2.5 fold in the kidney cortex of mice on low- Mg2+ diet and ~3.5 fold in MDCT epithelial cells culture in low Mg2+ medium. Under similar experimental conditions MMgT2 mRNA increases ~1.5 fold in kidney cortex and ~3 fold in MDCT 26

cells (Goytain and Quamme, 2008). We refer the interested audience to reviews by Schmitz et al., (2007), Bindels group (Alexander et al., 2008), and Quamme (2010) for a more in-depth elucidation of the specifics of TRPM6/7 channels and other Mg2+ entry mechanisms summarily described in this section. Exchangers While Mg2+ entry appears to be mediated by channels or channels-like mechanisms, Mg2+ extrusion is mediated by two mechanisms operating as exchangers. Based upon the electrochemical requirements favouring Mg2+ extrusion, these mechanisms are referred to as Na+-dependent and Na+-independent Mg2+ exchanger, respectively (Table 1). Because neither of these two mechanisms has been cloned, information about their operation, abundance and tissue specificity remains mostly circumstantial or indirect based upon experi- mental conditions or pharmacological inhibition. Na-dependent (Na+/Mg2+ Exchanger) The first evidence of the operation of a Mg2+ transport mechanism in mammalian cells was provided by Gunther et al., (1984). In this and a subsequent publication (Gunther and Vormann, 1985), these authors detailed the presence and operation of an amiloride-inhibited, Na+- dependent, Mg2+ extrusion mechanism in chicken red blood cells. This initial observation has been subsequently confirmed by other groups in mammalian red blood cells (Feray and Garay, 1986; Flatman and Smith, 1990; Xu and Willis, 1994) including human erythrocytes (Ludi and Schatzmannm 1987; Vormann et al., 1984; Raftos et al., 1999), and in a variety of mammalian cell types (see Romani and Scarpa, 2000 for a list). In addition, observation from Vormann and Gunther (Vormann and Gunther, 1987; Gunther and Vormann, 1992a), Wolf and collaborators (Wolf et al., 1996; Wolf et al., 1997), and our laboratory (Romani and Scarpa, 1990a; Romani and Scarpa 1990b; Romani et al., 1993a; Romani et al., 1993b; Fagan and Romani, 2000; Fagan and Romani, 2001; Cefaratti and Ruse, 2007; Cefaratti and Romani, 2007) has provided compelling evidence that this Na+-dependent, amiloride- inhibited Mg2+ extrusion mechanisms is specifically coupled to cAMP-production within the cells. From the experimental stand-point it is irrelevant whether cellular cAMP increases via

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stimulation of -adrenergic, glucagon, or PGE2 receptors, or via administration of forskolin or cell-permeant cyclic-AMP analogs. All these conditions, in fact, result in the activation of the Na+-dependent Mg2+ extrusion mechanism via phosphorylation. Conversely, pre-treatment of cells with inhibitors of adenylyl cyclase (e.g. Rp- cAMP) completely blocks Mg2+ mobilization irrespective of the modality utilized to enhance cAMP level (Wolf et al., 1997). Because the Mg2+ extrusion mediated by this exchanger strictly depends on the presence of a physiological concentration of Na+ in the extracellular milieu (Romani et al., 1993a; Fagan and Romani, 2000), it is generally accepted that the Na+-dependent Mg2+ extrusion mechanisms is a Na+/Mg2+ exchanger. As this Mg2+ extrusion mechanism has not been functionally cloned, we lack detailed information about its membrane abundance, structure, proximity to other cellular transporters with whom it may possibly interact, and stoichiometry. Early reports by Gunther and Vormann (1985) suggest the operation of electroneutral bases (2Na+in:1 Mg2+out) at least in chicken or turkey erythrocytes. This notion has not been confirmed in mammalian erythrocytes including human red blood cells, in which the exchanger appears to operate electrogenically on a 1Na+in:1 Mg2+out ratio (Ludi and Schatzmann, 1987; Flatman, 1990; Xu and Willis, 1994). The discrepancy between these reports is not apparent although it may depend on the experimental model (i.e. cell isolation vs cultured cells), composition of incubation medium, or modality of cellular Mg2+ loading. Irrespective of the stoichiometry of exchange and the exper- imental model, however, all the obtained results consistently indicate a Km for Na+ between 15 to 20 mM (Gunther, 1996; Tashiro and Konishi, 1997; Cefaratti et al., 1998). Pharmacological inhibition has done little to enhance our understanding of the modality of operation of the putative Na+/Mg2+ exchanger. Amiloride, imipramine and quinidine represent the three most widely utilized inhibitors of Na+-dependent Mg2+ extrusion (Gunther and Vormann, 1984; Feray and Garay, 1988; Gunther and Vormann, 1992a). However, because of their limited specificity, it is unclear as to whether they inhibit the Na+/Mg2+-exchanger directly, or indirectly by operating on other transport mechanisms including Na+ and K+ channel, ultimately altering

the cell membrane potential and the driving force for Mg2+ transport across the plasma membrane. Despite intense research, the Na+/Mg2+ exchanger is not cloned as yet. Work by Schweigel, Martens and colleagues (Schweigel et al., 2000) in mammalian rumen support the operation of a Na+/Mg2+ exchanger with kinetic parameters and characteristics similar to those described by other groups in a various mammalian cell types. Furthermore, by using a hybridoma screening procedure, this group has generated monoclonal antibodies against the Na+/Mg2+ exchanger present in porcine red blood cells. Incubation of sheep rumen epithelial cells in the presence of these antibodies has resulted in a significant inhibition of Mg2+ extrusion via this exchanger (Schweigel et al., 2000). Western blot analysis utilizing these antibodies has evidenced a protein band of ~70 KDa mr, which could tentatively correspond to the Na+/Mg2+ exchanger (Schweigel et al., 2000). This is the first time that information about the molecular size of the elusive Na+/Mg2+ exchanger is obtained, and the utilization of these antibodies could represent an ideal tool to identify and recognize this transporter in mammalian tissue. + Na -independent Under conditions in which no extracellular Na+ is available to exchange for intracellular Mg2+, an alternative Na+-independent Mg2+ extrusion mechanism becomes evident. The specificity of this transporter, however, is far from being characterized. Different cations, including Ca2+ or Mn2+, as well as anions (e.g. HCO3-, Cl-, or choline) (Gunther, 1993; Ebel et al., 2002) have been reported to be utilized by this mechanism to extrude Mg2+ from the cell. Hence, it remains unclear whether we are in the presence of distinct transport mechanisms, or in the presence of a transporter that can operate as an antiporter for cations or a synporter for cations and anions based upon the experimental conditions. Also, it is unclear whether the Na+-independent pathway is activated by hormonal stimulation. Results obtained in liver cells (Keenan et al., 1996; Fagan and Romani, 2000; Fagan and Romani, 2001) indicate that the stimulation by mix adrenergic agonists (e.g. epinephrine) elicit a Mg2+ extrusion that is equivalent to the sum of the amounts of Mg2+ mobilized by the separate stimulation of a1- and b-adrenergic receptors. More specifically, the 27

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selective stimulation of a1-adrenergic receptors by phenylephrine requires the presence of physiological concentrations of both Na+ and Ca2+ in the extracellular medium to elicit Mg2+ extrusion from liver cells (Fagan and Romani, 2000; Fagan and Romani, 2001). As phenylephrine stimulation appears to operate via Ca2+-CaM to induce Mg2+ extrusion, it is undefined whether this signalling pathway represents an alternative modality of activation of the Na+/Mg2+ exchanger, or it activates instead a different Mg2+ extrusion mechanism that can be reconciled at least in part with the aforementioned Na+-independent mechanism. Adding to the uncertainty, Ebel and collaborators (Ebel et al., 2002) have suggested that in red blood cells and hepatocytes the Na+-independent Mg2+ extrusion occurs via the choline transporter, which can be inhibited rather specifically by cinchona alkaloids. One controversial issue is whether the Na+- dependent and Na+-independent mechanisms operate as ATPases or require ATP for their operation. Reports by Gunther and collaborators (Gunther et al., 1990; Ebel et al., 2004) indicate a certain dependence of Na+-dependent Mg2+ extrusion on the presence of a physiological concentration of cellular ATP, the absence or decrease in cellular ATP content resulting in a reduced Mg2+ efflux from the cell (Gunther et al., 1990; Ebel et al., 2004). In the particular case of red blood cells, ATP and 2,3 bisphosphoglycerate both contribute to Mg2+ homeostasis and transport (Gunther et al., 1995). A regulatory effect of ATP on Mg2+ extrusion is not observed in purified liver plasma membrane vesicles (Cefaratti et al., 1998). It is true that no Mg2+ extrusion is observed in alkaline phosphatase- treated basolateral liver plasma membrane vesicles in the absence of ATP (Cefaratti and Romani, 2007), but this observation can reasonably be explained by the requirement of ATP to phosphorylate and activate the Na+/Mg2+ exchanger in the presence of PKA catalytic subunit (Cefaratti and Romani, 2007). Mg2+/H+ This exchange mechanism, originally identified in A. thaliana and termed AtMHX, appears to be present in all plants (Shaul et al., 1999). This transporter presents 11 putative transmembrane domains, is exclusively localized in the vacuolar 28

membrane of the plant, and electrogenically exchanges protons with Mg2+ or Zn2+. Interestingly, the ectopic overexpression of the transporter in tobacco plants sensitizes the plant to grow in the presence of elevated concentrations of Mg2+ (or Zn2+) (Shaul et al., 1999). Presently, no corresponding gene and transporter have been identified in mammalian cells, although evidence for a direct or indirect exchange of Mg2+ for H+ under certain conditions has been provided by Gunther (Gunther and Vormann, 1990a). An enhanced extrusion of cellular Mg2+ has been observed in cells incubated in an acidic extracellular environment, in which an inwardly oriented H+ gradient is imposed, provided that extracellular Na+ is present (Gunther and Vormann, 1990a; Dalal and Romani, 2010). Amiloride derivates that inhibit with high affinity the Na+/H+ exchanger are ineffective at blocking Mg2+ extrusion under these experimental conditions (Gunzel and Schlue, 1996), thus excluding that Mg2+ extrusion depends on the operation in reverse of the Na+/H+ exchanger in parallel with the forward operation of the Na+/Mg2+ exchanger. Carriers This section groups several novel Mg2+ transport mechanisms of murine or human origin identified as a result of diet restriction (i.e. Mg2+-deficient diet) or medium restriction (i.e. low extracellular Mg2+ content). Due to the limited information available and controversies in their modus operandi, these transport mechanisms are non- descriptively classified as carriers. SLC41 This family of Mg2+ transport mechanisms includes three members (A1, A2, and A3) that are distantly related to the prokaryotic MgtE channel identified by Maguire (Smith et al., 1995). We will discuss predominantly SLC41A1 and A2 isoforms since no study has addressed function and structure of the SLC41A3 isoform. SLC41A1 was the first member of this family to be identified (Wabakken et al., 2003). Based on the hydrophobic profile, this protein of ~56 kDa Mr was predicted to possess 10 transmembrane domains, two of which presented a discrete level of homology with MgtE (Wabakken et al., 2003). Northern blot analysis indicates a wide distribution of the SLC41A1 gene, but its

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abundance varies markedly among tissues, the highest expression being in heart and testis and the lowest being in hematopoietic tissues and cells (Wabakken et al., 2003). While modest under basal conditions, the expression of this gene is markedly up-regulated in the renal cortex of mice fed low Mg2+ diet for several days (Goytain and Quamme, 2005b). Functional expression of mouse SLC41A1 in X. oocyte indicates that this protein can transport Mg2+ but also Fe2+, Cu2+, Zn2+ and Cd2+ while Ca2+ is not transported nor inhibits Mg2+ transport (Goytain and Qaumme, 2005b). The original characterization of Mg2+ generated current, which would be tentatively consistent with SLC41A1 operating as a channel (Goytain and Qaumme, 2005b) or an electrogenic exchanger similar to the Na/Ca exchanger (Quednau et al., 2004), contrasts with a recent report by Kolisek et al., (2008). In this study the authors strongly advocate for SLC41A1 operating as a carrier and predominantly favouring Mg2+ efflux rather than influx. Following overexpression of SLC41A1 in HEK293 cells 1) no detectable Mg2+ currents is observed; 2) incubation of cells in Mg2+-free media results in a significant reduction of total Mg2+ content and [Mg2+]i; 3) the amplitude of Mg2+ loss depends on the number of SLC41A1 molecules expressed in the membrane and the induction time, and 4) the changes in [Mg2+]i are temperature sensitive but insensitive to the Mg2+ channel blocker CoHexamine (Kolisek et al., 2008). Furthermore, Kolisek and collaborators suggest that SLC41A1 forms high molecular weight complexes within the cell membrane with masses ranging from 1236 KDa to ~360 kDa, in stark contrast to the 56 KDa Mr of the monomer (Kolisek et al., 2008). Whether this observation indicates that the SLC41A1 monomer forms large multimeric complexes and/or interacts with auxiliary proteins is presently undefined. The reason for the absence of Mg2+-generated currents in this study as compared to the original observation by Goytain and Quamme (2005b) is not apparent. One possibility is that the murine (Goytain and Qaumme, 2005b) and human orthologs (Kolisek et al., 2008) operate differently. Based upon their high degree (>90%) of homology, the mouse and human SLC41A1 are expected to operate in a similar manner. Yet, the possibility that point mutations can dramatically alter SLC41A1 ion specificity and modality of function cannot be completely dismissed. In this

respect, it has to be noted that Goytain and Quamme (2005b) did not report a dependency of SLC41A1 operation on Na+ or other cations or anions following expression in X. oocytes, whereas Kolisek and collaborators (Kolisek et al., 2008) observed a marked Cl- conductance following expression in HEK293 cells, which was abolished by DIDS. Whether this reflects the operation of additional transport mechanisms or the presence of structural differences in the cell membrane of HEK293 cells as compared to X. oocyte are possibilities that need further investigation. Two additional SLC41 isoforms were identified in both humans and mice. SLC41A2 also transport Mg2+ as well as other divalent cations albeit with a different selectivity and inhibition profile than SLC41A1 (Goytain and Qaumme, 2005c). Aside from Mg2+, SLC41A2 can carry Ba2+, Ni2+, Co2+, Fe2+ and Mn2+ but not Ca2+, Cu2+ or Zn2+. At variance of SLC41A1, Mg2+ transport via SLC41A2 is inhibited by Ca2+ (Goytain and Qaumme, 2005c). Both SLC41A1 and A2 generate Mg2+ currents in X. oocyte, and the ionic uptake is voltage dependent with an apparent affinity of 0.75 mM and 0.31 mM, respectively (Goytain and Qaumme, 2005b; Goytain and Qaumme, 2005c). Also SLC41A2 is widely expressed in mammalian tissues, but the expression is not affected by low Mg2+ diet (Goytain and Qaumme, 2005c). At the structural level, SLC41A2 shares >70% homology with SLC41A1, and is also thought to have 10 trans- membrane domains, although this hypothesis is not supported by a recent study (Sahni et al., 2007) that instead suggests a structural arrangement of 2 x five trans-membrane spans linked together by a supplementary span motif. Hydrophobicity analysis indicates that the C- and N- termini are located on different sites of the cell membrane (Sahni et al., 2007), a configuration that will be consistent with a total of 11 trans-membrane segments. ACDP2 The human ACDP gene family was identified by Wang and collaborators (Wang et al., 2003a) as a possible candidate of the urofacial syndrome. Mapped to 10q23-10q24 chromosome, this gene family comprises 4 isoforms differentially located in human tissues. ACDP1 is essentially restricted to brain. ACDP2 is more widely expressed, but still retains the highest expression in brain while 29

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being absent in skeletal muscle. Both ACDP3 and ACDP4 are ubiquitous, but have the highest expression in the heart (Wang et al., 2003b). The murine distribution of ACDP isoforms is very similar to that observed in humans (Wang et al., 2004). Termed ancient conserved domain protein because all isoforms have one domain in common that appears to be phylogenetically conserved from bacteria to man (Wang et al., 2003a), these proteins share >50% homology to the CorC transporter, which together with CorB and CorD plays a role in Mg2+ efflux in pro- karyotes. Over-expression of ACDP2 in X. oocytes indicates that this protein can transport a broad range of divalent cations including Mg2+, Co2+, Mn2+, Sr2+, Ba2+, Cu2+, and Fe2+ while Zn2+ can inhibit its activity (Goytain and Quamme, 2005d). Mg2+ transport via ACDP2 is voltage dependent and occurs with a Km of ~0.5mM. The transport, however, operates independently of Na+ or Cl- ions (Goytain and Quamme, 2005d). As in the case of SLC41A1, the ACDP2 gene becomes over- expressed following a Mg2+ deficient diet (Goytain and Qaumme, 2005). NIPA Located in the SPG6 locus of chromosome 15q11- q13, the NIPA1 gene is so called for non- imprinted in Prader-Willi/Angelman syndrome, a disease characterized by a complex develop- mental and multisystem disorder (Butler, 1990). Located among about 30 genes linked to this disease (Butler, 1990), NIPA1 has also been implicated in autosomal dominant hereditary spastic paraplegia (HSP). The human and mouse genome contain four members of the NIPA family, termed NIPA1 trough NIPA4, with an overall similarity of ~40%. Homology between human and mice proteins is high at around 98%. Studies conducted by Goytain and Quamme indicate that both NIPA1 and NIPA2 (Goytain et al., 2007; Goytain et al., 2008a) can operate as Mg2+ transporter. Characterized by a sequence of 323 (NIPA1) and 359 amino acids (NIPA2) arranged to form 9 and 8 transmembrane spans, respectively, these two proteins can be distinguished based on their Km and specificity for Mg2+. While both proteins transport Mg2+ in a saturable fashion, NIPA1 has a Km for Mg2+ of ~0.66mM (Goytain et al., 2007) as compared to 0.31mM for NIPA2 (Goytain et al., 2008a). In addition, NIPA2 is highly specific for Mg2+ while NIPA1 can also transport Sr2+, Fe2+ or Co2+, albeit 30

to a lesser extent (Goytain et al., 2007). NIPA3, instead, transports Sr2+, Ba2+, Fe2+ and Cu2+ whereas NIPA4 transport Sr2+ and Ba2+. Interest- ingly, point mutations in NIPA1 (i.e. G100R or T45R) represent the basis for the insurgence of autosomal dominant HSP (Rainier et al., 2003). Both the glycine and threonine residues are conserved among ortholog NIPA1 channels in different species. There are no similar consensus sites in the paralogs NIPA2, NIPA3 and NIPA4, implying that the folding of these proteins might be different. Although NIPA2 appears to be normal in HSP patients, it cannot functionally replace NIPA1 to ameliorate HSP symptoms, nor can NIPA3 or NIPA4 substitute for the defective NIPA1. This is somewhat surprising for NIPA2 as the gene encoding for this protein is part of the 30 gene clusters associated with the Prade-Willi syndrome together with NIPA1. Presently, there is no information available as to whether the Prade-Willi syndrome presents alterations in Mg2+ homeostasis. Huntingtin The use of oligonucleotide microarray analysis to screen for Mg2+-regulated transcripts in epithelial cells indicates Huntingtin-interacting protein 14 (HIP14) and its related protein HIP14-like (HIP14L) as significantly (~ 3fold) upregulated by low-extracellular Mg2+ (Goytain et al., 2008b). Formed by approximately 532 amino acids organized in 6 transmembrane spans, HIP14 presents a strong sequence similarity to the ankyrin repeat protein Akr1p (Li and Li, 2004), and a 69% homology to HIP14L. In addition, HIP14 possesses a cytoplasmic DHHC cysteine- rich domain. Defined by the Asp-His-His-Cys sequence motif, this domain confers palmitoyl- acyltransferase activity to the protein and gives it the ability to palmitoylate membrane components and modulate their structure. Mg2+ accumulation via HIP14 and HIP14L appears to be electrogenic, voltage-dependent, and saturable with Km of ~0.87 and ~0.74mM, respectively (Goytain et al., 2008b). Inhibition of palmitoylation activity by 2- Br-palmitate or deletion of the DHHC domain decreases HIP14 mediated Mg2+ accumulation by ~50%, suggesting that palmitoylation is not required for basal Mg2+ transport. The widespread tissue distribution and intracellular localization of HIP14 (nuclear and perinuclear regions, Golgi complex, mitochondria, micro- tubules, endosomes, clathrin-coated and non-

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coated vesicles, and plasma membrane)(Yanai et al., 2006) has implicated this protein in numerous cellular processes including transcriptional regulation, mitochondrial bioenergetics, structural scaffolding, vesicle trafficking, endocytosis, and dendrite formation (Yanai et al., 2006). Its primary location, however, appears to be in the Golgi and post-Golgi vesicles (Yanai et al., 2006; Goytain et al., 2008b). Hence, it can be hypothesized that the ability of this protein to favour Mg2+ accumulation is linked to some extent to the role HIP14 plays in the physiological functioning of the compart-ments in which the protein is located. At the pathological level, the neuropathology of Huntington disease and the occurrence of progressive neurodegenerative disorders, cognitive deficits and choreic movements typical of this disease are linked to the abnormal expansion of glutamine residues from <34 to >37 at the 18th amino acid position (Li and Li, 2004). Presently, the mechanism responsible for the insurgence of these defects is unknown (Li and Li, 2004). Similarly unknown is whether the poly-glutamine expansion alters Mg2+ transport, and whether perturbation of Mg2+ homeostasis plays any role in the uprising of the neuronal defects typical of Huntington disease. Mg2+ transport in purified plasma membrane vesicles Because of the lack of functional cloning, several laboratories including ours have resorted to the use of plasma membrane vesicles to better characterize how different Mg2+ extrusion mechanisms operate in particular cell types. The plasma membrane model presents several advantages including: 1) the possibility to provide a well defined ionic extra- and intra-vesicular milieu composition to determine the modality of operation of the various Mg2+ transporters; and 2) the ability to investigate the operation of the different Mg2+ extrusion mechanisms in the absence of Mg2+ buffering by ATP, proteins or other cytosolic components, and partitioning within intracellular organelles. By purifying total liver plasma membrane or cardiac sarcolemmal vesicles as well as specific hepatic subpopulations enriched in basolateral or apical domains, our laboratory has been able to provide a better understanding of the selective location and specificity of the Na+- dependent and Na+-

independent Mg2+ extrusion mechanisms in liver cells and cardiac myocytes. The Na+-dependent extrusion mechanism located in the basolateral domain of the hepatocyte is selectively activated by Na+ (Cefaratti et al., 1998; Cefaratti et al., 2000), and specifically inhibited by imipramine (Cefaratti et al., 2000), but not amiloride and amiloride derivates (Cefaratti et al., 2000). Furthermore, the operation of the exchanger is completely inhibited by pre- treatment of basolateral vesicles with alkaline phosphatase, and restored by loading the vesicles with ATP and PKA catalytic subunit (Cefaratti and Ruse, 2007; Cefaratti and Romani, 2007), leaning further support to the notion that this exchange mechanism becomes operative upon phosphoryl- ation by cAMP. The Na+/Mg2+ exchanger continues to operate in the presence of zero trans Mg2+ across the plasma membrane (i.e., 20 mM Mg2+ inside and outside the vesicles), an indication that Mg2+ extrusion does not depend on the trans-membrane gradient for Mg2+ but rather on that of Na+, with a Km lower than 20mM (Cefaratti et al., 1998), in good agreement with kinetic data obtained in isolated hepatocytes (Romani et al., 1993b) and other cell types (Tashiro and Konishi, 1997). Experiments based on TPP+ distribution have confirmed the electro- genicity of this exchange mechanism in plasma membrane vesicles, and suggested a 1Na+in for 1Mg2+out exchange ratio under the majority of experimental conditions tested (Cefaratti et al., 1998; Cefaratti et al., 2000; Cefaratti and Ruse, 2007). Interestingly, removal of intravesicular Cl- switches the stoichiometric ratio of the exchanger from electrogenic to electroneutral (i.e. 2Na+in for 1Mg2+out) (Cefaratti and Romani, 2011). Moreover, in the presence of intra- vesicular Cl- an extrusion of ~35nmol Cl-/mg protein is observed within 1min from the addition of external Na+, in concomitance with the extrusion of Mg2+ and the accumulation of external Na+ into the vesicles (Cefaratti and Romani, 2011). Chloride ion extrusion is not inhibited by anion transport inhibitors like DNDS, DIDS, or niflumic acid, nor is it blocked by NKCC1 inhibitors like bumetanide or furosemide (Cefaratti and Romani, 2011), thus excluding that it occurs via one of these mechanisms. The only agent able to block the Cl- extrusion is imipramine (Cefaratti and Romani, 2011), w hich specifically 31

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blocks the Na+/Mg2+ exchanger operating in the basolateral domain of the hepatocyte (Cefaratti et al., 2000). Hence, it would appear that Cl- can be extruded either via the Na+/Mg2+ exchanger or via Cl- channels for partial charge compensation (Cefaratti and Romani, 2011). The possibility of a Cl- extrusion via the Na+/Mg2+ exchanger has been suggested by (Rasgado-Flores et al., 1994) in dialyzed squid axons, and it would also be in good agreement with the observation by Gunther and collab-orators that intracellular Cl- has a stimulatory role on the activity of the Na+/Mg2+ antiport in red blood cells (Ebel and Gunther, 2003). The basolateral domain of the hepatocyte is not the only site in which the operation of a Mg2+ extrusion mechanism has been observed. Experiments carried out in liver plasma membrane vesicles enriched in apical domain indicate the presence of two apparently distinct, unidirectional Mg2+ transport mechanisms, which extrude intravesicular Mg2+ for extravesicular Na+ and Ca2+, respectively (Cefaratti et al., 2000). This apical Na+-dependent Mg2+ transporter presents a Km for Na+ comparable to the baso- lateral transporter, and selectively uses Na+ over other monovalent cations in a manner similar to the basolateral exchanger. Like the basolateral antiport, this exchanger transports electro- genically 1Na+in:1Mg2+out (Cefaratti et al., 2000). From the pharmacological standpoint the apical and basolateral exchanger can be distinguished based on the specific inhibition of the apical exchanger by amiloride (Cefaratti et al., 2000), although it retains a significant level of sensitivity to imipramine inhibition, whereas only imipramine can block the basolateral antiport (Cefaratti et al., 2000). The apical exchanger can also be distinguished from the basolateral antiport based on its inability to operate in reverse mode (Cefaratti et al., 2000). The Ca2+-dependent Mg2+ extrusion mechanism is specifically located in the apical domain of the hepatocytes, is activated by micromolar Ca2+ concentration (Km<50M), and is insensitive to alkaline phosphatase pre-treatment (Cefaratti and Ruse, 2007; Cefaratti and Romani, 2007). The Mg2+ extrusion elicited by this antiport occurs on electro-neutral basis (i.e. 1Ca2+in:1Mg2+out) (Cefaratti and Ruse, 2007). The exchanger, 32

however, is not Ca2+ specific, as Mg2+ extrusion is observed following the extravesicular addition of micromolar concentrations of other divalent cations (Ca2+>>Co2+=Mn2+>Sr2+>>Ba2+>Cu2+>>Cd2+) (Cefaratti et al., 2000). Similarly to the apical Na+/Mg2+ antiport, the Ca2+-dependent mechanism is inhibited by amiloride or imipramine (Cefaratti et al., 2000). This observation raises the question as to whether we are in the presence of two distinct apical mechanisms, modulated by Na+ and cations, respectively. Several lines of evidence, however, do not fully support this possibility. First, the co- addition of Na+ and Ca2+ in purified apical plasma membrane vesicles subpopulations does not appear to significantly enlarge Mg2+ extrusion (Romani, personal observation). Second, amiloride inhibits both exchangers to a comparable extent at a similar concentration (Cefaratti et al., 2000). Third, alkaline phosphatase treatment does not affect the Mg2+ extrusion elicited by either exchanger in apical liver plasma membrane vesicles (Cefaratti and Ruse, 2007; Cefaratti and Romani, 2007). Fourth, neither of these exchangers can operate in reverse at variance of the basolateral Na+/Mg2+ antiport. Taken together, these observations suggest the operation of a non-selective exchange mechanism able to utilize monovalent or divalent cations to promote Mg2+ extrusion. At the present time, the physiological implication for the operation of such an exchanger in the apical domain of the hepatocyte is not fully clear. Circumstantial evidence, however, might support a possible role of Mg2+ in limiting Ca2+ sedimentation in the bile with consequent formation of bile stones (Moore, 1990). The operation of functionally similar Na+- and Ca2+-dependent Mg2+ extrusion mechanisms has also been observed in cardiac sarcolemma vesicles (Cefaratti and Romani, 2007). As in the case of liver plasma membrane vesicles, cardiac sarcolemma vesicles do not require intravesicular ATP for the operation of Mg2+ transporters (Cefaratti and Romani, 2007), and pretreatment of the vesicles with alkaline phosphatase specifically inhibits the Na+-dependent Mg2+ extrusion mechanism (Cefaratti and Romani, 2007). For technical reasons, it is presently unknown whether the Ca2+/Mg2+ exchanger in sarcolemmal vesicles can also utilize Na+ to promote Mg2+ extrusion.

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The operation of specific Mg2+ accumulation mechanisms has also been observed in plasma membrane vesicles from brush border cells of rabbit ileum (Juttner and Ebel, 1998) and from the duodenum and jejunum of rat (Baillien and Cogneau, 1995). By using membrane vesicles from rabbit ileum and cell permeant and non- permeant Mag-Fura, Juttner and Ebel have observed the operation of a saturable Mg2+ uptake mechanism when the intracellular Na+ concentration is higher than the extracellular one (Baillien and Cogneau, 1995). This process becomes inoperative when the Na+ gradient is reversed (i.e., [Na+]i<[Na+]o), the vesicles are in zero trans condition for Na+, or external Na+ is removed. At variance with the transporter observed in liver plasma membrane, the pathway in ileum vesicles is not reversible and appears to be electroneutral. Yet, it possesses a Km for Na+ of 16mM, a value similar to the Km calculated in liver plasma membranes (Cefaratti et al., 1998), in smooth muscle cells from guinea pig tenia caecum (Tashiro and Konishi, 1997), and in chicken erythrocytes (Schatzmann, 1993). Another similarity with the transporter operating in basolateral liver plasma membranes is the lack of inhibition by amiloride analogs. In good agreement with reports from Gunther and collaborators (Ebel and Gunther, 2003), this transporter is modulated by intravesicular anions, especially Cl- and SCN-, and markedly stimulated by antagonists of anion transport (e.g., H2-DIDS) (Juttner and Ebel, 1998). The main difference between plasma membrane vesicles from duodenum and jejunum (Baillien and Cogneau, 1995) is that a single Mg2+ uptake mechanism operates in the duodenum with a Km of 0.8mM, whereas two transporters operate in the jejunum with Km values of 0.15 and 2.4mM, respectively. In both these experimental models, Mg2+ but not Ca2+ accumulation is reduced in the presence of alkaline phosphatase inhibitors (Baillien et al., 2005), suggesting that Ca2+ and Mg2+ are transported via distinct pathways. This hypothesis is further supported by the observation that Mg2+ accumulation is inhibited by amiloride but not by Ca2+ channel antagonists. Consistent with the report by Juttner and Ebel (Juttner and Ebel, 1998), Mg2+ accumulation is stimulated by an intravesicular electronegative potential or an alkaline pHo (Baillien and Cogneau, 1995). The effect of external pH,

however, is lost when [Mg2+]o>1mM (Baillien and Cogneau, 1995). Under the latter condition, Mg2+ accumulation is enhanced by the presence of Na+ or K+ in the extravesicular space but is inhibited by the presence of divalent cations (Co2+>Mn2+>Ca2+>Ni2+>Ba2+>Sr2+) (Baillien and Cogneau, 1995). Regulation of Mg2+ transport and homeostasis While mammalian cells retains their basal Mg2+ content virtually unchanged under resting conditions, compelling evidence supports the ability of different hormones to induce the movement of large amount of Mg2+ in either direction across the eukaryotes cell membrane. As a result of these movements, changes in serum, total and to a lesser extent free Mg2+ content have been observed. Further, these changes have resulted in detectable variations in Mg2+ level within organelles, especially mito- chondria, with major repercussions on cellular bioenergetics. A full understanding of the physiological relevance of these changes in cellular Mg2+ content is far from complete. Yet, a picture is slowly emerging, which relates changes in total Mg2+ content to the utilization of metabolites (e.g. glucose) or to meaningful changes in Mg2+ content within discrete portions of the cell or cellular organelles, whereby variations in total Mg2+ content translate into changes in concentrations able to modulate the activity of specific enzymes located within these compartments. Mg2+ extrusion Several classes of hormones induce Mg2+ extrusion from various cell types or perfused tissues. For the most part, these hormones are catecholamine or hormones that increase cellular cAMP level by activating different GPCR receptors at the cell membrane level. The extrusion elicited by these agents affects to a varying extent the Mg2+ pools present within cytoplasm as well as within cellular compartments. The extrusion across the cell membrane primarily occurs via the Na+/Mg2+ exchanger previously described although a (partial) contribution of the Na+- independent pathway cannot be excluded. Magnesium extrusion can also be observed following metabolic treatments that decrease cellular ATP content, the main Mg2+ buffering component. Interestingly, several of the 33

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hormones that induce Mg2+ extrusion also elicit a glucose output from hepatocytes. Hence, it would appear that at least in this organ Mg2+ extrusion is functionally associated with glucose transport and utilization. Cyclic-AMP Dependent Extrusion Elliot and Rizack were the first to report an accumulation of Mg2+ in adipocytes stimulated by adreno-corticotrophic hormone in 1974, although they did not elucidate the modality of transport (Elliot and Rizack, 1974). The first extensive characterization of an hormonal effect on Mg2+ transport was provided by Maguire and colleagues in S49 lymphoma cells and primary lymphocytes stimulated by beta-adrenergic agonist or PGE1 (Bird and Maguire, 1978; Erdos and Maguire, 1980; Erdos and Maguire, 1983; Grubbs et al., 1984). Maguire and Erdos (1978) also provided the first observation that stimulation of protein kinase C enhanced Mg2+ influx in S49 cells whereas beta-adrenergic stimulation inhibited the process. Observation carried out in S49 cells lacking protein kinase A or adenylyl cyclase, however, indicated that cAMP was not mediating the inhibitory effect of beta-adrenergic agonists (Maguire and Erdos, 1978; Maguire and Erdos, 1980). At variance of what observed in primary lymphocytes (Wolf et al., 1997), Mg2+ transport in S49 cells appears to be independent of extra- cellular Na+ concentration or membrane potential (Grubbs and Maguire, unpublished observation). Further, Mg2+ turnover in S49 required more than 40 hours as compared to the much faster Ca2+ turn-over, which was accomplished in less than 3 hours (Grubbs et al., 1985). These initial observations were followed by a long series of report supporting the notion that b- adrenergic agonists and other hormones control Mg2+ homeostasis in mammalian cells. In the majority of eukaryotic cells, hormones or agents that increase cellular cAMP level elicit a significant extrusion of Mg2+ into the extracellular space or the circulation (Vormann and Gunther, 1987; Romani and Scarpa, 1990a; Romani and Scarpa, 1990b). This effect has been observed in cardiac ventricular myocytes (Vormann and Gunther, 1987; Romani and Scarpa, 1990a; Romani et al., 1993a; Howarth et al., 1994), liver cells (Romani and Scarpa, 1990b; Gunther et al., 1991; Romani et al., 1993b; Fagan and Romani, 2000; Fagan and Romani, 2001), red blood cells 34

(Matsuura et al., 1993), thymocytes (Gunther and Vormann, 1990b), and Erhlich ascites cells (Wolf et al., 1994) among other cells (see Romani and Scarpa, 2000 for a more comprehensive list), as well as in whole anesthetized animals (Gunther and Vormann, 1992; Keenan et al., 1995). In all cellular models, Mg2+ extrusion is a fast process that reaches the maximum within 8min from the application of the stimulus irrespective of the hormone (catecholamine, isoproterenol, glucagon, PGE1, or arachidonic acid) (Vormann and Gunther, 1987; Gunther and Vormann, 1990a; Romani and Scarpa, 1990a; Romani and Scarpa 1990b; Matsuura et al., 1993; Howarth et al., 1994; Wolf et al., 1994) or agent (i.e. forskolin or cell permeant cyclic AMP analogs) (Vormann and Gunther, 1987; Gunther and Vormann, 1990a; Romani and Scarpa, 1990a; Romani and Scarpa, 1990b; Matsuura et al., 1993; Romani et al., 1993b; Fagan and Romani 2000; Fagan and Romani, 2001) utilized to increase cellular cAMP level. The key role of cAMP in modulating Mg2+ extrusion is further emphasized by the observation that pretreatment with hormones or agents that either decrease cAMP production, such as carbachol (Romani and Scarpa, 1990a; Romani and Scarpa, 1990b; Romani et al., 1993b) and insulin (Romani et al., 2000), or prevent PKA activation (e.g. Rp-cAMP (Wolf et al., 1997)) inhibits cellular Mg2+ mobilization. In an open perfusion system, the amount of Mg2+ extruded from the organ returns towards baseline level within 8min from the application of the agonist irrespective of its dose or persistence in the perfusate (Romani and Scarpa, 1990a; Romani and Scarpa, 1990b), suggesting a rapid mobilization of Mg2+ from a well defined cellular pool that is rapidly depleted of its content. This notion is further supported by the evidence that submaximal doses of agonist sequentially infused within a few minutes from each other elicit Mg2+ extrusions of progressively decreasing amplitudes (Gunther and Vormann, 1990a). Under all these conditions, limited changes in cytosolic free [Mg2+]i are observed (Fatholahi et al., 2000; Amano et al., 2000), suggesting that Mg2+ is rapidly released from its binding and buffering sites, or form cellular organelle(s) and extruded across the cell membrane. Independent of the hormone utilized the cAMP-mediated Mg2+ extrusion occurs via the putative Na+/Mg2+ exchanger described previously. In fact, either the removal of extracellular Na+ (Gunther, 1996) or

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the presence of agents like amiloride (Vormann and Gunther, 1987; Gunther, 1996), which inhibits Na+ transport albeit in a non-specific manner, abolishes to a large extent the Mg2+ extrusion. Under either of these conditions, the amplitude of Mg2+ extrusion across the cell membrane is hampered and a more sustained rise in cytosolic free [Mg2+]i is observed (Fatholahi et al., 2000; Amano et al., 2000), supporting the notion that blocking Na+ transport prevents Mg2+ from being extruded across the cell membrane but not its released from binding/buffering sites and/or cellular pool(s) into the cytoplasm. Cyclic-AMP Independent Extrusion In 1989, Jakob and collaborators reported the first observation that phenylephrine can also elicit Mg2+ extrusion from liver cells via alpha1- adrenergic stimulation (Jakob et al., 1989). Subsequently, our laboratory (Keenan et al., 1996; Fagan and Romani, 2000) confirmed this observation and provided the first evidence that the stimulation of 1- and -adrenergic receptor are not alternative but rather additive and complementary processes in eliciting Mg2+ extrusion from liver cells, especially when the two classes of receptors are stimulated by mix adrenergic agonists such as epinephrine or norepinephrine (Keenan et al., 1996; Fagan and Romani, 2000). Pre-treatment with insulin only abolishes -adrenergic receptor mediated Mg2+ extrusion but leaves unaffected the Mg2+ mobilization mediated via 1-adrenergic receptors (Keenan et al., 1996). The inhibitory effect of insulin persists even in cells treated with cell- permeant cAMP analogs (Keenan et al., 1996). A similar inhibitory effect of insulin on b-adrenergic receptor mediated, cAMP-modulated Mg2+ extrusion has been observed in cardiac myocytes (Romani et al., 2000). This inhibition has been largely interpreted as the consequence of an inhibitory effect of insulin on the b-adrenergic receptor (Karoor et al., 1995) or a positive effect of the hormone on the cytosolic phospho- diesterase degrading cAMP (Smoake et al., 1995). A more recent report by Romero and collaborators (Ferreira et al., 2004), however, suggests that insulin can also modulate directly the Na+/Mg2+ exchanger, at least in red blood cells. Fagan and Romani (2000; 2001) further investigated the modality of Mg2+ extrusion

following 1-adrenergic receptor stimulation in liver cells. Their observation indicates that phenylephrine-induced Mg2+ extrusion strictly depends on the activation of capacitative Ca2+ entry (Fagan and Romani, 2001). Inhibition of IP3- induced Ca2+ release from the endoplasmic reticulum, chelation of cytosolic Ca2+, or inhibition of Ca2+ entry at the plasma membrane level all result in the complete inhibition of Mg2+ extrusion from the hepatocyte (Fagan and Romani, 2001). The scant information available about possible binding of Mg2+ by cellular proteins prevented the authors from ascertaining whether the extruded Mg2+ was mobilized from the ER or displaced from cytosolic binding sites following the massive entry of Ca2+ across the hepatocyte cell membrane (Fagan and Romani, 2001, and refs therein). Extracellular Na+ and Ca2+ are both required for the phenylephrine-induced Mg2+ extrusion to occur (Fagan and Romani, 2001). The absence of extracellular Ca2+, in fact, decreases the amplitude of Mg2+ extrusion by ~15% to 20% whereas extracellular Na+ is responsible for the remaining 80% to 85% of the extrusion. It is presently unclear whether Mg2+ extrusion occurs via the Ca2+-activated Na+- dependent mechanism observed in the apical domain of the hepatocyte, or whether Na+ is required to maintain membrane potential and facilitate Ca2+ entry across the hepatocyte cell membrane. It has to be noted, however, that in the absence of receptor activation, thapsigargin administration can mimic phenylephrine stimulation and elicit Mg2+ extrusion from the hepatocyte, even in the absence of extracellular Ca2+ (Fagan and Romani, 2001), although to a lesser extent. Hence, it would appear that an optimal level of cytosolic Ca2+ has to be attained in order for Mg2+ extrusion to occur via displacement from cellular binding sites or via a Ca2+-calmodulin-activated mechanism (Fagan and Romani, 2001). Mg2+ homeostasis and glucose The presence of redundant Mg2+ extrusion mechanisms or modalities of activation of a common Mg2+ extrusion pathway raises the question of the physiological significance of Mg2+ mobilization in mammalian cells. In the case of cardiac myocytes, an increase in extracellular Mg2+ level has been associated with a modulatory effect on the open probability of the L-type Ca2+- channels (Wang and Berlin, 2006) and a 35

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temporary decrease in SA node action potential (Howarth et al., 1994). In the case of liver cells, instead, Mg2+ transport has been associated with a regulatory role on glucose transport and utilization. Under conditions in which hormones like catecholamine (Keenan et al., 1996; Fagan and Romani, 2000), glucagon (Fagan and Romani, 2000), or phenylephrine (Keenan et al., 1996; Fagan and Romani, 2000) elicit Mg2+ extrusion from liver cells, a concomitant release of hepatic glucose, mostly via glycogenolysis, has been observed (Fagan and Romani, 2000). Interestingly, inhibition of Mg2+ extrusion by amiloride or imipramine also results in a marked inhibition of hepatic glucose output (Fagan and Romani, 2000). The converse is also true. Inhibition of glucose transport activity by phlorethin results in a qualitatively similar inhibition of Mg2+ extrusion from liver cells (Fagan and Romani, 2000). The presence of a close functional link between glucose and Mg2+ homeostasis is further emphasized by the observation that overnight starvation results in the complete depletion of hepatic glycogen and glucose as well as in a marked decrease (minus 15%) of total Mg2+ content as a consequence of the activation of the pro-glycemic hormones catecholamine and glucagon (Torres et al., 2005). Noteworthy, this decrease in hepatic Mg2+ content is equivalent to that elicited via in vitro stimulation of perfused livers by the same hormones (Torres et al., 2005), or that observed in the liver of type-I diabetic animals (Fagan et al., 2004), which are markedly decreased in cellular glycogen. The functional link between glucose and Mg2+ homeostasis is also observed under conditions in which glucose accumulation is stimulated by insulin or similar hormones in cardiac ventricular myocytes (Romani et al., 2000) or pancreatic beta cells (Henquin et al., 1983). In both experimental models, the amount of Mg2+ accumulated within the cells is directly proportional to the amplitude of glucose accum- ulation. Conversely, decreasing extracellular Mg2+ concentration directly reduces the amount of glucose accumulated within the cells (Fagan and Romani, 2000; Romani et al., 2000). Although indirect, a clear proof of the glucose/Mg2+ relationship is provided by diabetic conditions. Work by Alturas group (Resnick et al., 1993) and more recently by Resnick (Resnick, 1993) and Barbagallo (Barbagallo and Dominguez, 36

2007) indicate that cellular Mg2+ content is markedly decreased under type-I and type-II diabetes. Originally observed in red blood cells (Resnick et al., 1993; Resnick, 1993; Barbagallo and Dominguez, 2007), the decrease has also been reported in various other tissues including muscles, liver (Fagan et al., 2004), and cardiac myocytes (Reed et al., 2008). Interestingly, Mg2+ extrusion via -adrenergic signalling remains operative and is actually up-regulated in liver cells from diabetic animals (Fagan et al., 2004) while it is markedly inhibited in cardiac myocytes from the same animals (Reed et al., 2008). Whether this reflects a differential operation and modulation of 2-adrenergic receptors in liver cells vs 1-adrenergic receptors in cardiac cells is not completely clear. Both cell models show a marked inhibition of the Mg2+ entry mechanism(s), which persists also in liver plasma membrane vesicles (Fagan et al., 2004; Cefaratti et al., 2004). Addition of glucose or glycogen to plasma membrane vesicles from diabetic animals renormalizes the amplitude of Mg2+ extrusion, but is ineffective at restoring Mg2+ accumulation in vesicles (Cefaratti et al., 2004). The defect in total cellular Mg2+ content appears to be strongly associated with the decrease in protein synthesis and ATP production detected in the cells (Reed et al., 2008). Supplementation of exogenous insulin restores both these parameters as well as Mg2+ homeostasis and extrusion provided that insulin is administered for at least two weeks (reed et al., 2008). As indicated previously, it appears that the role of insulin in modulating Mg2+ homeostasis is not restricted to controlling glucose homeostasis and accumulation or the release of pro-glycemic hormones like glucagon, but extends to a direct modulation of the Na+/Mg2+ exchanger (Ferreira et al., 2004). The latter effect would not only directly increase cellular Mg2+ content but it could also reverberate on the insulin receptor itself. Data obtained in animals maintained on low Mg2+ diet indicate that a decrease in cellular Mg2+ content affects the ability of the insulin receptor to properly phosphorylate the downstream insulin receptor substrate (IRS) and propagate the signalling within muscle cells (Suarez et al., 1995). This result might be of relevance in explaining the decrease in glucose accumulation observed in skeletal muscles under diabetic conditions (Suarez et al., 1995).

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Mg2+ homeostasis and ATP Hormonal stimuli represent the most dynamic modality by which a cell can rapidly extrude 10% to 15% of its total cellular Mg2+ content within a few minutes from the application of the agonist. Mg2+, however, can also be extruded following the treatment with various agents that impact cellular ATP content and production. Cyanide (Harman et al., 1990; Dalal and Romani, 2010), mitochondrial uncouplers (Akerman, 1981; Kubota et al., 2005), fructose (Gaussin et al., 1997), ethanol (Tessman and Romani, 1998), or hypoxia (Gasbarrini et al., 1992) are just some of the agents whose addition impact cellular Mg2+ homeostasis. All these agents, in fact, have in common that they decrease cellular ATP content by either preventing the mitochondrial electron chain from generating ATP at various levels (cyanide or uncouplers), or by acting as an ATP trap (fructose), or by altering the redox state of pyridine nucleotide within the mitochondrion or the cell (ethanol). As ATP represents the major buffering component for Mg2+ within the cell (Scarpa and Brinley, 1981; Luthi et al., 1999), a decrease in this phosphonucleotide moiety results in an increase in cytosolic free [Mg2+]i, and ultimately in a detectable extrusion from the cell (Harman et al., 1990; Gasbarrini et al., 1992; Gaussin et al., 1997; Tessman and Romani, 1998; Dalal and Romani, 2010). The Mg2+ extrusion can be observed to a larger extent in erythrocytes, which possess limited cellular buffering capacity and no compartmentation (Hwa et al., 1993), but it can also be observed in cells that present additional buffering by proteins or cellular organelles in addition to phosphonucleotides (Harman et al., 1990; Gasbarrini et al., 1992; Gaussin et al., 1997; Tessman and Romani, 1998; Dalal and Romani, 2010). In the case of fructose, the changes in cytosolic Mg2+ have been associated with an activation of the glycogen phosphorylase, which ultimately results in glycogenolysis activation and glucose utilization to restore ATP levels (Gaussin et al., 1997). In the majority of these experimental conditions, the increase in cytosolic free [Mg2+]i is usually modest, and considerably lower than the increase expected to occur based upon the corresponding decrease in ATP level, which strongly supports the notion that Mg2+ is for the most part extruded from the cell. Furthermore, as ATP level decreases as a result of mitochondria poisoning or changes in pyridine nucleotide ratio,

it would appear that not phosphorylation but the rise in cytosolic Mg2+ albeit modest is sufficient to activate the Mg2+ extrusion mechanism and limit the rise in cytosolic free Mg2+ concentration to approximately 100-200 mM at the most (Harman et al., 1999). Cellular ATP plays a key role in regulating Mg2+ extrusion. Evidence for this role has been provided in the giant squid axon (Di Polo and Beauge, 1988) as well as in mammalian hepatocytes (Gunther and Hollriegl, 1993) or erythrocytes (Gunther et al., 1995). In squid axon, the Na+-dependent Mg2+ extrusion requires a physiological level of ATP. As the phospho- nucleotide level decreases, so does the amplitude of extrusion (Gunther and Hollriegl, 1993). In erythrocytes and hepatocytes, instead, ATP appears to regulate the Na+-independent Mg2+ extrusion process (Gunther and Hollriegl, 1993; Gunther et al., 1995). The exact role of ATP in regulating the process, however, is unclear as it does not appear that the extrusion process is mediated by an ATPase mechanism. This notion is supported by the observation that a decrease in cellular ATP level (as it occurs for example under diabetic or alcoholic conditions) paradoxically results in an increased extrusion of Mg2+ via the Na+-dependent mechanism in a manner directly proportional to the decrease in ATP level (Tessman and Romani, 1998; Fagan et al., 2004). Hence, it appears that the role of ATP is predominantly that of a ligand for Mg2+ both in the cytoplasm and the mitochondrial matrix (Scarpa and Brinley, 1981; Luthi et al., 1999), and that a decrease in ATP results in an increase in free Mg2+ and its consequent extrusion from the cell. Mg2+ accumulation The identification of several Mg2+ entry mechanisms strongly support the hypothesis that cellular Mg2+ is dynamically maintained through the operation of entry and exit mechanisms that are differentially regulated by hormones and metabolic conditions. A striking difference is there, however, between the Mg2+ exit and the Mg2+ entry mechanisms. In the case of Mg2+ extrusion mechanisms we have a good understanding of the signalling activating these mechanisms but we lack any structural information about the mechanisms themselves. In the case of Mg2+ entry mechanisms, instead, 37

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we do have structural information about several of these mechanisms but for the most part we lack detailed information about their individual activation by hormones or second messengers, and their possible cooperation under specific conditions. Role of Protein Kinase C Experimental evidence indicates that mammalian cells can accumulate large amounts of Mg2+ as a result of hormonal stimulation. Hormones like carbachol, vasopressin, angiotensin-II, or insulin have been indicated as able to either inhibit cAMP-mediated Mg2+ extrusion or reverse the extrusion into Mg2+ accumulation in various cell types (Romani and Scarpa, 1990a; Romani et al., 2000). The list of cells that respond to hormonal stimulation by accumulating Mg2+ is rather long [see Romani and Scarpa, 2000 for a list], and involve all kind of cells: cardiac myocytes (Romani and Scarpa, 1990a; Romani et al., 2000), smooth muscle cells (Touyz and Schiffrin, 1996), hepatocytes (Romani and Scarpa, 1990b; Romani et al., 1992), platelets (Hwang et al., 1993), lymphocytes (Grubbs and Maguire, 1986), fibroblasts (Ishijima and Tatibana, 1994), and pancreatic beta cells (Henquin et al., 1983), just to name a few. In addition to inhibiting cAMP production, several of the hormones indicated above can activate protein kinase C (PKC) as part of their cellular signalling. Evidence supporting a role of PKC in mediating Mg2+ accumulation has been provided by several laboratories. Maguire and collaborators have indicated that adminis- tration of phorbol-myristate acetate (PMA), which directly activates PKC, elicits a marked accumulation of Mg2+ in S49 lymphoma cells (Erdos and Maguire, 1983). A similar effect of PMA has been reported by Somogyis group in thymocytes (Csermely et al., 1987), and by our laboratory in cardiac myocytes (Romani et al., 1992) and hepatocytes (Romani et al., 1992). Furthermore, our group has reported that down- regulation of PKC by exposure to a large dose of PMA for 3 hours completely abolishes the ability of cardiac and liver cells to accumulate Mg2+ while leaving unaffected the responsiveness of these cells to adrenergic agonists (Romani et al., 1992). An inhibitory effect has also been observed following administration of the PKC inhibitors calphostin (Touyz and Schiffrin, 1996) or staurosporine (Gunther and Vormann, 1995). Alteration in PKC distribution and activity and 38

defective accumulation of Mg2+ has been observed in arterial smooth muscle cells (Yang et al., 2001), hepatocytes of animals exposed to alcohol (Torres et al., 2010), and in liver cells of diabetic animals (Tang et al., 1993). Protein kinase C activation is only part of the integral response of hormones like angiotensin-II or vasopressin. The interaction of these hormones with their receptor, in fact, activates phospholipase C which, in turn hydrolyses PIP2 to generate diacylglycerol (DAG) and IP3. In turn, these two molecules activate protein kinase C and IP3 receptor in the ER, respectively. Activation of the latter receptor results in a marked but transient increase in cytosolic Ca2+, and in a more sustained entry of Ca2+ through the capacitative Ca2+ entry mechanism. Hence, Ca2+ signalling is an integral component of the cellular response elicited by these hormones. Yet, the contribution of this second messenger in mediating Mg2+ accumulation is poorly defined. Liver cells loaded with Bapta-AM, which effectively chelates cytosolic Ca2+, are unable to extrude and accumulate Mg2+ following stimulation by phenylephrine and PMA, respectively (Romani et al., 1993b). The artificial increase in cytosolic Ca2+ elicited by thapsigargin administration also prevents Mg2+ accumulation (Romani et al., 1993b) and actually induces a Mg2+ extrusion from the liver cell if applied for more than 3-5 min (Romani et al., 1993b; Fagan and Romani, 2001). Because of the different time-scale and amplitude of the changes in cellular Ca2+ and Mg2+ content (Romani et al., 1993b), it is difficult to properly correlate these experimental variations. Cytosolic free Ca2+ transiently increases several orders of magnitude while cytosolic free Mg2+, which is already in the millimolar or submillimolar range, increases by ~10-15% (Fatholahi et al., 2000) at the most, although in absolute terms this amount is far larger than the overall change in cytosolic Ca2+ mass. An unresolved point of inconsistency in the role of Ca2+ and PKC signalling in regulating Mg2+ accumulation is provided by the reports that the administration of phenylephrine, which activates PKC signalling in addition to the inositol 1,4,5 trisphosphate/Ca2+ pathway, does not elicit Mg2+ accumulation but induces a Mg2+ extrusion from liver cells (Fagan and Romani, 2001). This point

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raises the question as to what modulates the differential response of the cell to the administration of phenylephrine or vasopressin. One possibility could be that different PKC isoforms are activated under one condition and not the other. For example, hepatocytes possess 3 classical and at least 2 novel PKC isoforms (Tang et al., 1993). Thus, it is reasonable to envisage that one isoform (or class of isoforms) is involved in mediating Mg2+ accumulation whereas another isoform (or class of isoforms) is involved in modulating Mg2+ extrusion. Consistent with this hypothesis, recent data from our laboratory suggests that PKCe is essential for Mg2+ accumulation to occur (Torres et al., 2010). Under conditions in which the expression of this isoform is inhibited by antisense or its translocation to the cell membrane is prevented for example by ethanol administration, no Mg2+ accumulation can be detected in liver cells (Torres et al., 2010). Interestingly, this PKC isoform has the highest affinity for Mg2+ among all PKC isoenzymes, with a Km of ~1mM (Konno et al., 1989), close to the physiological free [Mg2+]i measured in the cytoplasm of the hepatocyte (Corkey et al., 1986; Fatholahi et al., 2000) and other mammalian cells as well (Touyz and Schiffrin, 1996). Although the mechanism ultimately responsible for Mg2+ accumulation into the hepatocyte has not been identified, it is worth considering the recent observation by Bindels and collaborators that in the absence of PKC activation or following RACK1 over-expression, RACK1 can bind to TRPM6, and possibly TRPM7, at the level of the kinase domain and inhibit the channel activity (Cao et al., 2008). Role of MAPKs These results mentioned above, however, do not exclude the possibility that additional signalling pathways (e.g. MAPKS) are involved in determining the differing response of the hepatocyte under apparently similar stimulatory conditions. In agreement with previous reports from Alturas group (Yang et al., 2000) and Touyzs laboratory (Touyz and Yao, 2003), our laboratory has evidenced that pharmacological inhibition of ERK1/2 and p38 MAPKs in liver cells abolishes PKC mediated Mg2+ accumulation (Torres et al., 2006). In this respect, it has to be noted that the inhibition of MAPKs hampers Mg2+ accumulation in vascular smooth muscle cells, and significantly affect cyclin activity (Touyz and Yao, 2003) and consequently the ability of the

cells to progress in the cell cycle (Touyz and Yao, 2003). This effect may occur via changes in nuclear functions directly regulated by Mg2+, as proposed by Rubin (2005) and/or via changes in nuclear functions of ERK2, which depends on Mg2+ level to properly dimerize, translocate and activate specific nuclear targets (Waas and Dalby, 2003). The role of ERK1/2 in Mg2+ regulation is further emphasized by the recent report that increased phosphorylation of ERK1/2 and increased expression of TRPM6 has been observed upon EGF administration to renal epithelial cells (Ikari et al., 2008; Ikari et al., 2010). The role of these MAPKs, however, is far from being elucidated as this kinase appears to be involved to some extent in also mediating Mg2+ extrusion (Kim et al., 2005; Torres et al., 2006). Role of EGF As mentioned, direct and indirect evidence suggests a key role of EGF in modulating Mg2+ accumulation, at least in kidney cells. The administration of EGF controls proper operation of TRPM6 in the apical domain of renal epithelial cells to promote Mg2+ accumulation (Ikari et al., 2008; Thebault et al., 2009; Ikari et al., 2010). Point mutations in EGF sequence result in limited function of TRPM6, and limited or no Mg2+ accumulation within the cells (van der Wjist et al., 2009). The modulation of TRPM6 appears to occur via MAPKs signalling, most likely ERK1/2 coupled to activator protein-1 (AP-1) (Ikari et al., 2010). Indirect evidence of EGF regulation is provided by the evidence that antibodies against EGF used in several forms of colon cancer (Cunningham et al., 2004; Dimke et al., 2010) induce Mg2+ wasting and hypomagnesaemia. Serum Mg2+ level and Mg2+ sensing mechanism The experimental evidence that mammalian cells accumulate or extrude Mg2+ under a variety of experimental conditions suggests the presence of a sensor for the cytosolic Mg2+ concentration, whereby the cell would operate accordingly either extruding the excess cation or accumulating it to restore the set-point. Compelling evidence for the presence of such a sensor mechanism is provided by the observation that prolonged exposure to 0mM [Mg2+]o decreases cytosolic free Mg2+ concentration by approximately 50% in cardiac ventricular 39

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myocytes (Quamme and Rabkin, 1990), MDKC (Quamme and Dai, 1990), or MDCT cells (Dai et al., 1997). The new cytosolic Mg2+ level is maintained as long as the cells are incubated in the presence of 0 mM [Mg2+]o, but returns to the normal level as soon as [Mg2+]o is increased (Quamme and Rabkin, 1990; Quamme and Dai, 1990; Dai et al., 1997), in a frame of time that is directly proportional to the extracellular Mg2+ concentration utilized (Quamme and Rabkin, 1990; Quamme and Dai, 1990; Dai et al., 1997). The process is prevented by the presence of the L-type Ca2+-channel inhibitors verapamil or nifedipine, or La3+ in the extracellular milieu (Quamme and Rabkin, 1990). The absence of significant changes in cytosolic free Ca2+ concentration under these experimental conditions suggests a direct effect of these inhibitory agents on the Mg2+ entry mechanism (Quamme and Rabkin, 1990). This observation led the authors to propose the operation of a specific Mg2+ channel in these cells, anticipating the identification of TRPM6 (Schlingmann et al., 2002) and TRPM7 (Nadler et al., 2001) as specific Mg2+ channels. Mg2+ accumulation is favoured by ion re- distribution. In fact, renal epithelial cells accumulate Mg2+ as a result of phosphate (Dai et al., 1997) or potassium (Dai et al., 1991) redistribution across the cell membrane. The dependence of Mg2+ accumulation on K+ redistribution across the plasma membrane suggests that Mg2+ transport is the result of changes in membrane potential, possibly for charge compensation (Schweigel et al., 1999; Tashiro et al., 2002; Schweigel and Martens, 2003). In this respect, it is interesting to note that pathological conditions characterized by a marked decrease in tissue Mg2+ content (e.g., diabetes) (Fagan et al., 2004) are also characterized by an inability to properly transport potassium (Mondon et al., 1974; Taylor and Agius, 1988). Whether the effect on K+ occurs through changes in membrane potential, or indirectly via a reduced operation of the Na+/K+-ATPase coupled to the operation in reverse of the Na+/Mg2+ exchanger (Gunther and Vormann, 1995), is a matter for future investigation. Humans and many mammals present a circulating Mg2+ level of ~1.2-1.4 mEq/L (Geigy, 1984; Mudge, 1989). Clinical and experimental evidence 40

indicates that serum Mg2+ level decreases in humans and animals in several chronic diseases (Fagan et al., 2004). Yet, there is a remarkable lack of information as to whether serum Mg2+ undergoes circadian fluctuations following hormonal or non-hormonal stimuli (e.g., fasting or exercise). The infusion of catecholamine in conscious humans (Joborn et al., 1985; Bailly et al., 1990) or ovine (Rayssiguier, 1977), or the administration of isoproterenol (Guideri, 1992) or epinephrine in the presence of 1-adrenoceptor blockade (Rayssiguier, 1977) have provided contrasting results. More recent results, however, would indicate that isoproterenol infusion elicits in a marked dose- and time- dependent increase in circulating Mg2+ content (Gunther and Vormann, 1992; Keenan et al., 1995). The increase is serum Mg2+ is maximal within 20min from the agent administration (Keenan et al., 1995), and remains unchanged up to 2 hours even following the removal of the agonist (Keenan et al., 1995). This time frame indicates that the increase in serum Mg2+ is not dependent on the increase in heart rate and the decrease in mean arterial pressure elicited by the -adrenergic agonist (Keenan et al., 1995). In fact, the infusion of sodium nitroprusside, which mimics the decrease in mean arterial pressure induced by isoproterenol, has no effect on serum Mg2+ levels (Keenan et al., 1995). The persistence of an elevated serum Mg2+ level for up to 2 hours also implies that operation of secondary and not fully elucidated mechanisms activated by the stimulation of -adrenergic receptor. Consistent with the larger distribution of 2 versus 1 adrenergic receptors in the body (Molinoff, 1984; Barnes, 1995), the increase in serum Mg2+ occurs via the specific activation of 2-adrenergic receptor, as it is mimicked by the administration of the selective 2-adrenergic agonist salbutamol, and it is inhibited by the specific 2-blocker ICI- 118551 (Keenan et al., 1995). Due to the amplitude of the increase in Mg2+ level into the bloodstream, it is reasonable to envisage that the adrenergic agonists are mobilizing Mg2+ from more than one tissue (Keenan et al., 1995). The involvement of bone has been proposed by Gunther and co-workers (Gunther and Vormann, 1992) based on the observation that the infusion of carbonic anhydrase inhibitor prevents isoproterenol-mediated Mg2+ mobilization into the blood of anesthetized rats. Based upon the pre-infusion level of serum Mg2+, the glomerular

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filtration rate (1.62 mL/min), and the fractional excretion (17%) (Shafik and Quamme, 1989), changes in renal excretion do not appear to contribute significantly to the increase in serum Mg2+ level, at least during the first two hours following adrenergic agonist infusion. It is interesting to note, however, that hormones that increase plasma Mg2+ by mobilizing the cation from different organs or tissues usually increase Mg2+ resorption in the Henles loop, de facto preventing any net loss of Mg2+. The inconsistency of changes in serum Mg2+ level observed in the literature, however, does not have a clear explanation. Several factors may contribute to this incongruity, including the proportion of b-adrenergic receptor subtypes in different experimental models, the ability of adrenergic agonists to stimulate with various potency different adrenergic receptors subtypes, and the modality, rate and duration of drug infusion. This inconsistency has also hampered our ability to assign a physiological significance to the eventual increase in serum Mg2+ level following catecholamine infusion. The observation that serum Mg2+ level increases under certain conditions may imply that specific organs or tissues have the ability to sense these changes and act accordingly. Presently, no specific Mg2+ sensing mechanism has been identified. However, the Ca2+ sensing receptor (Brown et al., 1993) can senses changes in circulating Mg2+ level in a range of concentrations far higher than those of Ca2+ (Nemeth and Scarpa, 1987) but consistent with the increase in serum Mg2+ level reported in the literature (Gunther and Vormann, 1992; Keenan et al., 1995). The observation that in distal convoluted tubule cells (MDCT) of the mouse the Ca2+-sensing receptor can be activated with comparable sensitivity by both extracellular Ca2+ and Mg2+ (Bapty et al., 1998a) suggests interesting hypotheses in terms of whole body physiology. The activation of this sensor mechanism would inhibit glucagon- or vasopressin-mediated entry of Mg2+ into the cell (Bapty et al., 1998b) and favour the elimination of the cation with the urine. This would then explain the clinical and experimental evidence that hypermagnesemia and hypercalcemia inhibit hormone-stimulated cAMP-mediated resorption of Mg2+ and Ca2+ along the different segments of the nephron (Quamme and Dirks, 1980). Also, it would represent a distal regulatory mechanism to

restore magnesemia to a physiological level following the increase observed in anesthetized animals infused with adrenergic agonists (Gunther and Vormann, 1992; Keenan et al., 1995). Whether this sensing mechanism and/or its associated modulatory components are altered under diabetic conditions in which an increased magnesuria is observed it remains to be elucidated. At variance of an increase in calcemia, which is associated with muscle weakness and arrhythmia, an increase in serum Mg2+ level appears to be well tolerated in vivo. Rats infused with boluses of Mg2+ that increase serum Mg2+ level by 50% do not exhibit significant systemic hemodynamic changes but show a marked increase in coronary artery flow (Dipette et al., 1987). Baboons infused with pharmacological doses of Mg2+ sufficient to prevent epinephrine- induced cardiac arrhythmias show a significant attenuation of the epinephrine-induced increase in mean arterial pressure and systemic vascular resistance (Stanbury, 1948). It would appear, therefore, that an elevated level of [Mg2+]o can regulate catecholamine release from peripheral and adrenal sources (James et al., 1988) and modulate cardiac contractility (Howarth et al., 1994). Taken together, these observations suggest that an increase in serum Mg2+ level following adrenergic stimulation can: 1) act as a feed-back mechanism to modulate catechol- amine release and activity; and 2) contribute to improved blood flow and O2 delivery to the heart and possibly other tissues at a time when an increase in energy production is expected. Physiological role of intracellular Mg2+ The conclusions presented in the previous sections strongly suggest a key role for Mg2+ as an indispensable component for enzymes, phospho- metabolites, and channel activity (Grubbs and Maguire, 1987; Romani and Scarpa, 1992). Several glycolytic enzymes, including hexokinase, phosphofructokinase, phosphoglycerate mutase, phosphoglycerate kinase, enolase and pyruvate kinase, show activation at low and inhibition at high Mg2+ concentrations (Otto et al., 1974; Garfinkel and Garfinkel, 1988). The adenylate cyclase is another example of an enzyme directly regulated by Mg2+ (reviewed in Maguire, 1984). All these processes occur at Mg2+ concentrations 41

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between 0.5 to 1mM, which are well within the fluctuations in free [Mg2+]i measured in the cytoplasm of various cells including hepatocyte (Corkey et al., 1986). With the exception of the glycolytic enzymes, studies attempting to evidence a regulatory role of Mg2+ for cytosolic enzymes have been disappointing, mostly because of the underlying assumption that Mg2+ would operate as Ca2+ in modulating enzyme activity. At variance from Ca2+, Mg2+ concen- tration in the cytoplasm and extracellular fluids is in the millimolar or submillimolar range. Consequently, an increase or decrease in cytosolic Mg2+ level equivalent to the changes observed for Ca2+ will go largely undetected by the common fluorescent or 31P-NMR techniques. Heretofore, a role of Mg2+ as transient regulator of cytosolic enzymes appears to be unlikely. It has to be noted that even under conditions in which hormonal and non-hormonal stimuli elicit major fluxes of Mg2+ across the cell plasma membrane in either direction, massive translocations of Mg2+ that increase or decrease the total cellular Mg2+ content by 1 to 2mM (or 5-10% of total cell content) result in minor or no changes in cytosolic free [Mg2+]i (Harman et al., 1990; Romani et al., 2000). This disconnect can be explained by assuming that the source or destination of transported Mg2+ is a cellular compartment or organelle, or a major binding site, or that Mg2+ is highly buffered. Hence, regulation of cellular functions by Mg2+ should not be necessarily expected to occur in the cytosol but within organelles, and in the plasma, where Mg2+ concentration can rapidly increase or decrease more than 20% (Gunther and Vormann, 1992; Keenan et al., 1995). In the following pages we will highlight what is known about regulatory effects of extracellular or intracellular Mg2+ on the operation of cation channels in the plasma membrane, as well as on mitochondria respiration and volume following changes in Mg2+ concentration within the organelle. Ca2+- and K+-channels The first report of a regulatory effect of intracellular free Mg2+ on calcium channels was by White and Hartzell (1988). These authors reported that increasing intracellular free [Mg2+]i from 0.3 to 3.0mM by internal perfusion had a dual effect in cardiac ventricular myocytes. It had 42

a small effect on basal L-type Ca2+-channels current (ICa) but decreased by more than 50% the amplitude of ICa elevated by cAMP-dependent phosphorylation. The effect of Mg2+ was not due to changes in cAMP concentration or in the velocity of phosphorylation but appeared to be a direct effect on the phosphorylated channel or on the channel dephosphorylation rate (White and Hartzell, 1988). Similar results were also reported by Agus and Morad (1991) in guinea pig cardiac myocytes. The block induced by Mg2+on the Ca2+ current depended on a direct effect on the inactivation state of the channel as the block persisted in the presence and in the absence of cAMP and was not reversed by elevation of extracellular Ca2+ concentration or addition of catecholamine (Agus and Morad, 1991). The effect of Mg2+ on Ca2+-channels is not restricted to cardiac cells or to an intra-cellular site of action. Bara and Guiet-Bara (2001) have shown that in vascular smooth muscle cells and endothelial cells in human placenta, MgCl2 (and to a lesser extent MgSO4) regulates the influx of Ca2+ through voltage-gated Ca2+ channels by acting at an extracellular site on the channel, thus modulating the tonus of the vessels. Evidence for a similar block by extracellular Mg2+ on T-type Ca2+-channels has been provided by Serrano et al., (2000). The modulatory effect of Mg2+ appears to take place at the EF-hand motif of the COOH-terminus of Cav1.2 channels as recently evidence by Catterall and his group (Brunet et al., 2006). Extracellular Mg2+ also modulates the activity of store-operated Ca2+ channels. Studies in intact, pressurized rat mesenteric arteries with myogenic tone indicate that 10 mM extracellular Mg2+ can mimic nifedipine in preventing or reversing the vasoconstriction elicited by phenylephrine administration, but not that induced by K+ depolarization (Zhang et al., 2002). Therefore, Mg2+ abolishes the vasoconstriction attributed to Ca2+ entry through store-operated channels, which are activated following phenylephrine administration, contributing to maintain both myogenic tone and 1-adrenoceptor-induced tonic vasoconstriction. These data may have direct implication to explain some of the modifications that occur under hypertensive conditions, in which a decrease in plasma Mg2+ has often been reported.

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Intracellular Mg2+ also affects he operation of store-operated calcium release-activated Ca2+ (CRAC) channels (Prakriya and Lewis, 2002). CRAC channels are highly Ca2+-selective under physiological ionic conditions whereas removal of extracellular divalent cations makes them freely permeable to monovalent cations, in particular Na+. Experimental evidence indicates that intra- cellular Mg2+ can modulate the activity and selectivity of these channels therefore affecting monovalent cation permeability. While an effect of intracellular Mg2+ on CRAC channels cannot be completely excluded, a report by Prakriya and Lewis (2002) suggests that the channels modulated by intracellular Mg2+ are not CRAC channels, but a different class of channels that open when Mg2+ is washed out of the cytosol. These channels have therefore been termed Mg2+-inhibited cation (MIC) channels, as they present distinctive functional parameters in terms of inhibition, regulation, ion permeation and selectivity (Prakriya and Lewis, 2002). Potassium channels are also targets for Mg2+. Matsuda (1991) has reported that the presence of Mg2+ on the cytoplasmic side of the inwardly rectifying K+ channel blocks the outward currents without affecting the inward currents. The Mg2+ block is achieved at a half-saturation concentration of 1.7M. When the Mg2+ concentration is elevated to 2-10M, the outward current fluctuated between two intermediate sublevels in addition to the fully open and closed configuration. However, these concentrations of Mg2+ are far from being physiological and it is difficult to envision the occurrence of a similar regulatory effect under normal conditions without invoking Mg2+ microcompartmentation. A regulatory role of intracellular Mg2+ on Kv channels in vascular smooth muscle cells has been observed by Tammaro et al., (2005). These authors have observed that an increase in intracellular Mg2+ in a range of concentrations consistent with its physiological variations can slow down the kinetic of activation of the Kv channel, cause inward rectification at positive membrane potentials, and shift the voltage-dependent inactivation (Tammaro et al., 2005). Overall, this represents a novel mechanism for the regulation of this channel in the vasculature. Intracellular Mg2+ also modulates large-conductance (BK type) Ca2+- dependent K+ channels by either blocking the

pore of BK channels in a voltage-dependent manner, or by activating the channels independ- ently of changes in Ca2+ and voltage by preferentially binding to the channel open conformation at a site different from Ca2+ sites. Interestingly, Mg2+ may also bind to Ca2+ sites and competitively inhibit Ca2+-dependent activation (Shi et al., 2002). The inhibitory effect of Mg2+ is not restricted to cell membrane channels. Experimental evidence by Bednarczyk et al., (2005) indicates that Mg2+ in the mitochondrial matrix can modulate gating and conductance of mitochondrial KATP channels, which play a key role under ischemia/reperfusion conditions. Mitochondrial dehydrogenases Mitochondria represent one of the major cellular pools for Mg2+, its concentration being 14 to 16 mM (Gunther, 1986). Evidence from this (Romani et al., 1991) and other laboratories (Zhang and Melvin, 1992; Zhang and Melvin, 1996; Kubota et al., 2005) suggests that Mg2+ can be mobilized from mitochondria under various conditions including hormone-mediated increase in cytosolic cAMP level through a mechanism(s) that has not been fully elucidated but it appears to involve the adenine nucleotide translocase (Romani et al., 1991). Several reviews have analyzed in detail how Mg2+ homeostasis is regulated in the organelle (Flatman, 1984; Gunther, 1986; Ogoma et al., 1992), and we refer to those reviews for further information. This section will focus on some recent evidence about a role of intra- and extra-mitochondrial Mg2+ on the activity of specific mitochondrial proteins. It is commonly accepted that changes in matrix Ca2+ can affect the rate of mitochondrial de- hydrogenases and therefore the rate of respiration (McCormack et al., 1990; Hansford, 1994). Yet, limited evidence supports a similar role for Mg2+ although the activity of several mitochondrial dehydrogenases has been observed to increase within minutes from the application of hormonal or metabolic stimuli despite the absence of a detectable increase in mitochondrial Ca2+ (Moravec and Bond, 1991; Moravec and Bond, 1992). The role of matrix Mg2+ as regulator of dehydrogenases and consequently mitochondrial respiration has been investigated by measuring the activity of several dehydrogenases in 43

Intracellular Mg homeostasis

Chapter 2

mitochondria under conditions in which matrix Ca2+ and/or Mg2+ concentration were varied. From those data, it appears that -ketoglutarate dehydrogenase and pyruvate dehydrogenase are not regulated by changes in mitochondrial Mg2+, whereas Mg2+ removal increases several fold the activity of succinate and glutamate dehydro- genases (Panov and Scarpa, 1996a; Panov and Scarpa, 1996b). This evidence would therefore indicate that changes in matrix Mg2+ content (in combination with or in alternative to changes in mitochondrial Ca2+) can control mitochondria respiration, at least under well defined conditions. In this respect, mitochondrial Mg2+ content appears to change quite significantly during transition from state 3 to state 4 (Brierley et al., 1987), affecting at the same time the amplitude of mitochondria respiration. In addition, data from our laboratory (Romani et al., 1991), Zhang and Melvin (1996), and Kubota et al (2005) would suggest that mitochondrial Mg2+ can be mobilized by catecholamine stimulation via cAMP. Hence, the enhanced mitochondrial respiration elicited by catecholamine could depend, at least in part, on cAMP-mediated modulation of mitochondrial Mg2+, which, in turn, will stimulate directly some dehydrogenases while rendering others more susceptible to the Ca2+ concentrations present in the mitochondrial matrix. The regulatory effect of Mg2+ on mitochondrial function is not restricted to the dehydrogenases activity but affects also the anion channel present in the mitochondrial membrane (Beavis and Powers, 2004) as well as the opening of the permeability transition pore (Dolder et al., 2003). The mitochondrial inner membrane anion channel (IMAC) transports various anions, and is involved in regulating the organelle volume in conjunction with the K+/H+ antiporter. Although its fine regulation is not completely elucidated as yet, experimental evidence suggests that matrix Mg2+ and protons inhibit IMAC, maintaining the channel in its closed state (Beavis and Powers, 2004). Kinetic studies by Beavis and collaborators further support a main role of Mg2+ in maintaining the channel in a condition that would allow fine modulation by small changes in pH and proton distribution under physiological conditions (Beavis and Powers, 2004). 44

The permeability transition pore (PTP) is a proteinaceous pore that opens in the inner mitochondrial membrane following a marked decrease in membrane potential and results in the rapid equilibration and redistribution of matrix and extramitochondrial solutes down their concentrations gradient. While it is still debated exactly which proteins participate in the pore formation and how the pore opens, it is well established that an increase in mitochondrial Ca2+ content facilitates the opening whereas an increase in mitochondrial Mg2+ antagonizes it. This effect can be appreciated well in yeasts, which do not possess a canonical PTP (Bradshaw and Pfeiffer, 2006). According to one model of regulation, creatine kinase can regulate the opening of the permeability transition pore by tightly associating to the mitochondrial membrane and remaining in an active state (Dolder et al., 2003). Both these processes are Mg2+-dependent, and Mg2+ removal from the extramitochondrial environment results in a decline in creatine kinas activity and in the opening of the permeability transition pore Dolder et al., 2003). Considering the effect of Mg2+ on mitochondrial function and channels (see previous section), it appears that Mg2+ plays more than one role within this organelle regulating: 1) mitochondrial volume; 2) ion composition; 3) ATP production; and 4) metabolic interaction with the hosting cell. Reticular G6Pase The Endoplasmic Reticulum (ER) represents one of the major cellular Mg2+ pools, with a total concentration of 14 to 18mM (Romani and Scarpa, 1992). Yet, limited information is available about any major role of luminal Mg2+ on reticular functions other than protein synthesis (Rubin, 2005). It is also unknown whether Mg2+ is buffered or chelated by endoluminal proteins in the same way that Ca2+ is complexed. Furthermore, we do not have any information about the transport mechanism(s) that allow(s) ER (and SR) to attain and maintain such a large luminal Mg2+ concentration. Work by Volpe and collaborators (Volpe et al., 1990; Volpe and Vezu, 1993), Gusev and Niggli (2008), and Laver and Honen (2008) suggests that cytosolic and perhaps luminal Mg2+ concentration can have a major effect in limiting Ca2+ uptake

Magnesium in the Central Nervous System

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into the ER/SR and its release from the organelle via IP3 (Volpe et al., 1990) and ryanodine receptor (Laver and Honen, 2008). In the latter case, a direct effect of Mg2+ on the receptor has been observed (Gusev and Niggli, 2008; Laver and Honen, 2008). Less clear is whether a similar effect takes also place at the level of the IP3 receptor. More recently, our laboratory has reported that cytosolic Mg2+ can have a regulatory role on the activity of reticular glucose 6-phosphatase (G6Pase) in liver cells (Doleh and Romani, 2007). This effect is biphasic, with an optimal stimulatory effect at ~0.5mM [Mg2+]i and an inhibitory effect at higher concentrations (Doleh and Romani, 2007). The effect of Mg2+ appears to take place at the level of the G6Pi transport component of the G6Pase enzymatic complex in that it can be inhibited by EDTA (as Mg2+ chelating agent) or taurocholic acid, which permeabilizes the ER membrane and allows G6Pi to bypass the transport mechanism and be delivered directly to the catalytic site of the G6Pase within the ER lumen (Doleh and Romani, 2007). Investigation is in progress to determine whether Mg2+ exerts a similar modulatory effect on other ER enzymes. Cell pH and volume As mentioned earlier, exposure of the cell to cyanide (Dalal and Romani, 2010), fructose (Gaussin et al., 1997), hypoxia (Harman et al., 1990; Gasbarrini et al., 1992), ethanol (Tessman and Romani, 1998), or choline chloride (Romani et al., 1993b) results in a marked cellular acidification associated with a decrease in cellular ATP content and a major Mg2+ extrusion. This extrusion is the consequence of a decrease in buffering capacity (ATP loss) as well as binding affinity. Now, a report by Yamaguchi and Ishikawa (2008) highlights a new and important regulatory role of intracellular Mg2+ on the operation of the electrogenic Na+-HCO3- co- transporter NBCe1-B in a range of concentrations that are consistent with the variations in Mg2+ content measured within the cytoplasm of various cell types (Grubbs and Maguire, 1987). This regulatory effect is exerted by Mg2+ and not by MgATP, and requires a functional N-terminus on the NBCe1-B transporter (Yamaguchi and Ishikawa, 2008). It is still unclear whether Mg2+ binds the N-terminus of the transporter directly or exerts its effects via an intermediate, Mg2+- modulated regulatory protein (Yamaguchi and

Ishikawa, 2008). At a cytosolic [Mg2+] of ~1mM (a physiological Mg2+ concentration measured in the cytosol of various cells (Corkey et al., 1986; Grubbs and Maguire, 1987), the NBCe1-B current is inhibited by ~50%, and no detectable current can be measured for free Mg2+ concentrations of 3-5mM. More or less at the same time, the group of Hirano and collaborators reported that increasing cellular Mg2+ content has a stimulatory role on the expression of aquaporin 3 in CaCo-3 cells (Okahira et al., 2008). This isoform of aquaporin is highly expressed in the gastro-intestinal tract, in which it absorbs water, glycerol and urea. The effect of Mg2+ on aquaporin mRNA expression appears to involve cAMP/PKA/CREB signalling, as well as we MEK1/2 and MSK1 (Okahira et al., 2008), suggesting short- and long-term regulation on the activity and expression of this protein. It is presently undefined whether Mg2+ exerts a similar regulatory role on other aquaporin isoforms. Moreover, as aquaporin 3 is also expressed in brain, erythrocytes, kidney, and skin, a modulatory role of Mg2+ on the protein expression in these tissues may have major relevance for various physiological and/or pathological conditions. Taken together, these two sets of information suggest that Mg2+ can have a major regulatory role on cellular pH, cellular volume, and cellular cation concentration, especially Na+. Also, as aquaporin 3 mediates glycerol and urea accumulation, possible repercussions on fatty acid metabolism and urea cycle must be taken into proper consideration. Cell cycle Cell cycle (Maguire, 1988; Sgambato et al., 1999; Touyz et Yao, 2003), cell proliferation (Wolf et al., 2009b) and cell differentiation (Covacci et al., 1998; Wolf et al., 1998; Di Francesco et al., 1998) have all been associated with the maintenance of an optimal cellular Mg2+ level. Under conditions in which cellular Mg2+ accessibility is restricted or reduced, cell proliferation and cell cycle progression are markedly impaired. A decrease in extracellular Mg2+ content also affects cell differentiation (Covacci et al., 1998; Wolf et al., 1998; Di Francesco et al., 1998). The mechanisms by which a decrease in cellular Mg2+ content affects these cellular processes have been 45

Intracellular Mg homeostasis

Chapter 2

attributed to defective MAPKs (Touyz and Yao, 2003) and p27 (Sgambato et al., 1999) signalling, increased oxidative stress level (Wolf et al., 2009b), and decreased MgATP levels (Di Francesco et al., 1998; Rubin, 2005). Because cellular MgATP level is at a level optimal for protein synthesis (Rubin, 2005), any alteration in this metabolic parameter will have major repercussion on the proper functioning of the cell. In addition, extracellular Mg2+ levels regulate integrins signalling, de facto modulating the interaction among cells and between cells and extracellular matrix (Trache et al., 2010). Hence, consistent with the long-term regulatory function of Mg2+ proposed by Grubbs and Maguire (1987) several years ago, all these observations underlie the important role of Mg2+ to guarantee cell cycle progression and retention of proper cell morphology a nd f unction, a voiding u ndesired References
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progression towards cell death or neoplastic destiny (Wolf et al., 2009a). Conclusions In the last few years, our understanding of cellular and whole body Mg2+ homeostasis has significantly advanced. While it still lags behind as compared to the knowledge available for other ions such as Ca2+, H+, K+ or Na+, the identification of putative Mg2+ channels and transport mechanisms in the membrane of the cell or mitochondria, as well as an improved understanding of the signalling pathways and conditions regulating Mg2+ transport are providing new tools to address essential questions about the physiological role Mg2+ plays inside the cell and in the whole body.


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Magnesium transport across the blood-brain barriers


Mounir N. Ghabriel and Robert Vink
Discipline of Anatomy and Pathology & Adelaide Centre for Neuroscience Research, School of Medical Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.
mounir.ghabriel@adelaide.edu.au

Abstract The finding that magnesium levels are reduced in acute and chronic brain diseases has led to a recent surge in interest in the role of magnesium in the normal and injured nervous system, although the mechanisms of magnesium decline in pathological conditions and its availability in the neural tissue after administration are not fully understood. The brain has two main barrier systems: (1) the blood-brain barrier (BBB) formed by brain capillary endothelial cells which separate the blood from the extracellular fluid in the neuropil; and (2) the blood-CSF barrier (BCSFB) formed by choroidal epithelial cells which separate the blood from the CSF. Genetic studies in families with hereditary hypomagnesemia have identified mutations in two genes encoding claudin-16 (paracellin-1) and claudin-19, both localized at tight junctions between nephron epithelial cells and providing passive paracellular conductance for magnesium in the kidney. Endothelial cells of the BBB also express claudins, although whether members of the claudin family expressed at the BBB and BCSFB have similar conductance for magnesium akin to the role of claudin-16 and -19 in the nephron remains to be confirmed. Recently, the transient receptor potential melastatin (TRPM) members TRPM6 and TRPM7 have been identified as cation channels for magnesium transport. Although it is not known if choroidal epithelial cells express TRPM6 and TRPM7, these molecules are expressed by brain endothelial cells and may play a role in magnesium transport. While it is evident that magnesium enters the CNS through the BBB and is actively transported by choroidal epithelial cells into the CSF, the mechanisms of its entry into the brain will require further investigation.


Introduction Magnesium is a critical cation and an essential nutrient for normal body functions; hence mechanisms exist in the body for its homeostasis through a highly integrated feedback system involving intestinal absorption, renal excretion, bone metabolism and the parathyroid gland (Dai et al., 2001). Magnesium is involved in a myriad of biochemical processes including acting as a cofactor in the activation of many intracellular enzymes (Aikawa, 1976; Ebel and Gunther, 1980; Gunther, 2008) and is important for protein synthesis (Terasaki and Rubin 1985) and cell membrane stabilization (Bara and Guiet-Bara 1984). Magnesium deficiency reduces protein synthesis, serum antibody activity and the immune response (McCoy and Kenney, 1975), and induces CNS epileptiform activity (Morris, 1992) and hypomagnesemic tetany (Fontenot et al., 1989). There is an inverse correlation between dietary magnesium and the level of C- reactive protein (a marker of inflammation) and the level of E-selectin (a marker of endothelial cell dysfunction) (Song et al., 2007). Recent technical developments have allowed better assessment of the levels of magnesium in the body, although the blood, which is used in routine testing, contains only 0.3% of the total body magnesium (Elin, 1987). Approximately one half of the magnesium contained in the body is stored in the bone, while the rest exists in the soft tissues, mainly in the intracellular compartment, with less that 1% being present in the extracellular compartment (Elin, 1988). Indeed, magnesium is the second most common cation in the intracellular compartment after potassium. More than one half of the plasma content of magnesium is bound to plasma proteins, and the remaining free ionized magnesium (Mg2+), the metabolically active fraction, is held within a narrow range (0.53- 0.67mM) in normal healthy controls (Altura and Altura, 1991). The recom-mended daily allowance 59

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ranges from 320 to 420mg/day for women and men, respectively (Bergman et al., 2009). There has been a recent surge in interest in the dynamics and role of magnesium in the normal and injured brain due to accumulating evidence of a reduction in the level of total and free Mg2+ in the brain in acute and chronic neurological diseases (Vink et al., 1987; 2009). In intensive care patients with traumatic brain injury (TBI) reduction of serum ionized Mg2+ correlates with the severity of TBI as determined by the Glasgow Coma Scale Score (Kahraman et al., 2003). In experimental TBI in the rat there is a sustained decline in intracellular Mg2+, detected by phosphorus magnetic resonance spectroscopy, that correlates with motor deficit (Cernak et al., 2004; Heath and Vink, 1996), while magnesium administration significantly improves motor outcome (Heath and Vink 1999; Turner et al., 2004). Magnesium administration reduces TBI- induced brain edema (Feldman et al., 1996) and restores blood-brain barrier (BBB) effectiveness to Evans blue tracer when compared to injured non-treated rats (Esen et al., 2003; Imer et al., 2009). Magnesium also reduces brain edema after cold-induced brain injury (Turkoglu et al., 2008). However, the mechanisms of magnesium decline in pathological conditions and its availability in the neural tissue after administration are still unclear (Vink et al., 2009). Therapeutically, magnesium can be administered orally, intravenously or intramuscularly (Elin, 1988). It is most commonly used used as magnesium chloride, sulphate, gluconate, acetate (Fine et al., 1991) or lactate (Fine et al., 1991; Simoes Fernandes et al., 1985). Magnesium therapy has been utilized in numerous experimental and clinical settings including migraine (Peikert et al., 1996), asthma (Cheuk et al., 2005), depression (Eby and Eby, 2006), anxiety (Kara et al., 2002), diabetes (Resnick et al., 1993; Wester and Dyckner, 1987), hypertension (Wester and Dyckner, 1987), atrial fibrillation (Fanning et al., 1991), sleep disorders, insomnia and chronic fatigue (Takahashi et al., 1992), dementia (Glick, 1990), osteoporosis (Sojka and Weaver, 1995), fibromyalgia (Porter et al., 2010), pain (Soave et al., 2009), eclampsia (Euser and Cipolla, 2009), constipation (Guerrera et al., 2009), cerebral palsy (Rouse, 2009), lacunar stroke (Muir et al., 2004), TBI (McIntosh et al., 60

1988) and aneurysmal subarachnoid hemorrhage (van den Bergh, 2009; Yahia et al., 2005). The current review will briefly outline the status of magnesium in the body, then will summarize current knowledge of its distribution in the CNS and discuss its transport across barrier membranes in the brain compared to its transport in other organs such as the kidney. Magnesium absorption and excretion Much of our knowledge about magnesium absorption and excretion was gained in the latter part of the twentieth century and particularly in the nineties. Studies of normal subjects showed that magnesium bioavailability from high magnesium containing food sources such as almonds is equal to that obtained from soluble magnesium acetate, but enteric coating of magnesium chloride impairs magnesium bioavailability (Fine et al., 1991). Magnesium absorption occurs mainly in the small intestine (Brannan et al., 1976; Graham et al., 1960; Schroeder et al., 1969). The kinetics and rate of magnesium absorption are not dependant on calcium intake (Brannan et al., 1976). Under basal conditions the small intestine absorbs 3050% of the magnesium intake, although this percentage diminishes with senescence, chronic renal disease and increasing intake (Musso 2009). The fractional magnesium absorption appears to fall progressively so that absorption as a function of intake is curvilinear (Fine et al., 1991). The fecal magnesium appears to be primarily derived from material that is not absorbed by the body rather than magnesium secreted by the intestine (Aikawa, 1976). Approximately 80% of the absorbed magnesium passes in the glomerular filtrate (Dai et al., 2001; Quamme and de Rouffignac, 2000). More that 95% of the magnesium filtered by the glomerulus is reclaimed mainly in the thick ascending loop of Henle (60-70%) and to a lesser extent (10-15%) in the proximal convoluted tubules (Brunette et al., 1974; Dai et al., 2001; Quamme 1997; Quamme and de Rouffignac, 2000). A further 10% of magnesium in the filtrate is claimed in the distal convoluted tubule (de Rouffignac and Quamme, 1994) and this segment contributes to magnesium conservation (Quamme 1997). Magnesium reabsorption within the thick

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ascending loop appears to be passive and occurs via the paracellular route (de Rouffignac and Quamme, 1994), being driven by the trans- epithelial voltage (Quamme, 1997; Quamme and de Rouffignac, 2000), where positive luminal charge favors movement of magnesium from the luminal to the abluminal side of nephron epithelium (Quamme, 1997). On the other hand magnesium transport in the distal convoluted tubule is active and transcellular (Quamme, 1 997). While the mechanisms of magnesium transport were unclear and speculative (Dai et al., 2001; Quamme and Dirks, 1980) more recent

reports point to specific paracellular (Efrati et al., 2005; Simon et al., 1999) and transcellular (Hoenderop and Bindels, 2005) routes across barrier membranes. Barriers between the blood and the CNS Two main fluid compartments are associated with the brain, the extracellular fluid (ECF) that bathes neurons and glial cells, and the cerebrospinal fluid (CSF) located in the subarachnoid space and ventricles of the brain (Figure 1).

Figure 1. Diagram showing locations of the blood-brain barrier (BBB), the blood-CSF barrier (BCSFB) and the meninges. The cranial dura mater (DM) encloses all intracranial contents, and is formed of two fused layers that separate at certain places to enclose dural venous sinuses, such as the superior sagittal sinus (SSS). The subdural space (SDS) is a potential space as the dura and arachnoid mater (AM) are closely apposed. The arachnoid mater is impermeable to CSF contained in the subarachnoid space (SAS). The arachnoid mater forms the arachnoid granulations (AG) that pierce the dural wall of the superior sagittal sinus and drain the CSF into the venous blood. Brian capillaries (BC) are lined with endothelial cells joined by tight junctions and form the BBB, thus they control the composition of the extracellular fluid (ECF). Ventricles (V) of the brain contain CSF produced mainly by the choroidal epithelium, which covers the choroid plexuses (CP) and form the BCSFB. The ventricles are lined with ependymal cells (E), which are continuous with the choroidal epithelium at the neck of the choroid plexus. The core of the choroid plexus is formed of pia mater and contains fenestrated capillaries. CSF leaves the ventricular system via the three foramina of the fourth ventricle, represented here by two channels (F). The CSF and ECF of the brain equilibrate through the pia mater on the surface of the brain, and the ependyma lining the ventricles (double-headed arrows). 61

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Exchange between the blood and these fluid compartments can potentially occur at four sites, which have barrier mechanisms: (1) brain capillary endothelial cells, which separate the ECF from the blood and form the BBB; (2) the choroidal epithelial cells at the ventricular surface of the choroid plexuses, which interface between the blood and the CSF and form the blood-CSF barrier (BCSFB); (3) the arachnoid mater, which surrounds the CSF contained within the subarachnoid space; (4) the circumventricular organs of the brain, which have fenestrated capillaries. The four sites form potential entry routes for water, molecules, electrolytes, toxins, pathogens and drugs (Segal, 2000). The arachnoid mater and circumventricular organs The arachnoid mater forms an external barrier, the morphological substrates of which are the flat tightly packed mesothelial cells (the arachnoid barrier cell layer) with their numerous tight junctions. They separate the CSF from the subdural space and the dura (Fig.1), which has fenestrated capillaries (Nabeshima et al., 1975; Vandenabeele et al., 1996). The arachnoid is avascular (Alcolado et al., 1988) and, relative to brain capillaries, has a much less surface area, thus its contribution to transport from the subdural space to the CSF and brain is negligible. Also the circumventricular organs of the brain, although they have fenestrated capillaries (McKinley et al., 2003; Sunn et al., 2003), they are provided with an internal mechanism that contributes to a barrier between these sites and the surrounding brain. They have rapid venous return systems, which compensates for the leaky capillaries, effectively preventing the spread of marker molecules to the surrounding brain tissue (Hashimoto, 1988; Segal, 2000). Therefore, the two main routes that are likely to have greater involvement in the regulation of the ECF and CSF environments are the BBB and BCSFB (Abbott et al., 2010). The blood-brain barrier (BBB) The concept of a BBB has been proposed over a century ago (Bradbury 1979) and is still being developed (Wolburg et al., 2009). The BBB is formed by endothelial cells of brain capillaries, the characteristics of which are influenced by the surrounding microenvironment, including astro- cytes and pericytes (Wolburg et al., 2009). Apart 62

from capillaries in the circumventricular organs which have fenestrated endothelial cells, those in the rest of the brain are lined with specialized endothelial cells which show few endocytotic vesicles, have no fenestrations and are rich in mitochondria (Brightman and Reese, 1969; Fenstermacher et al., 1988; Reese and Karnovsky, 1967; Rubin and Staddon, 1999; Sedlakova et al., 1999). Brain endothelial cells have high electrical resistance (Butt et al., 1990) and are joined by tight junctions (TJs) (Begley and Brightman, 2003; Rubin and Staddon, 1999). These TJs have long been recognized as the sites of exclusion of protein tracers as detected by electron microscopy (Brightman and Reese 1969; Reese and Karnovsky 1967; Sedlakova et al., 1999). Freeze-fracture electron microscopy revealed that these tight junctions have an elaborate and complex arrangement of network of inter- connecting strands of intramembranous particles, that particularly cleave with the P-face leaving corresponding grooves on the E-face of replicas of the split membrane (Sedlakova et al., 1999). The BBB therefore plays a pivotal role in the tight regulation and rigorous stabilization of the chemical composition of brain ECF against fluctuations in the plasma chemistry, thus promoting normal neuronal signaling. The privileged status of the brain being protected by the BBB has a downside, in restricting access by drugs and therapeutic agents into the brain when needed (Abbott and Romero, 1996). Although the barrier nature of the BBB has been emphasized, it is obviously a selective barrier, as it should allow passage of nutrients and electrolytes. Thus endothelial cells of brain vessels are endowed with a host of receptors, enzymes and carriers in a polarized distribution to promote selective transport (Abbott et al., 2010; Betz et al., 1980; Ohtsuki and Terasaki, 2007; Roberts et al., 2008). Brain endothelial cells express the glucose transporter Glut-1 protein (Dick et al., 1984; Harik et al., 1994; Ohtsuki and Terasaki, 2007; Pardridge et al., 1990; Pardridge, 1991), which is coupled to neuronal demand (Leybaert, 2005) and its expression increases in hypoxia (Harik et al., 1994). Rat brain endothelial cells express a barrier antigen (Sternberger and Sternberger, 1987), neutralization of which leads to reversible opening of the BBB to exogenous and endogenous proteins (Ghabriel et al., 2000; 2004). Lipophilic molecules can penetrate

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endothelial cell membranes by diffusion, although some of these may be effluxed via ABC transporters, such as P-glyco-protein (Begley 2004; Miller, 2010; Shen and Zhang, 2010). Macromolecules may cross the BBB via receptor- mediated transcytosis (Descamps et al., 1996), or adsorptive-mediated transcytosis (Villegas and Broadwell, 1993; Zlokovic et al., 1990). Water- soluble nutrients and metabolites can to a limited degree pass passively across the BBB, but greater bulks of water, hydrophilic molecules and electrolytes have to be transported via carrier systems and channels (Zhang et al., 2002). Amino acids, nucleosides, small peptides, organic anions and organic cations are transported via solute carriers (Abbott et al., 2010; Koepsell et al., 2003; Ohtsuki and Terasaki, 2007). Unlike in other organs, such as the intestinal wall where the extracellular space is large and is occupied by loose connective tissue, that of the brain is very narrow as neuronal and glial membranes are closely apposed leaving a gap of 20-50 nm (Vanharreveld et al., 1965) with a small volume of ECF. A small change in the ECF volume may lead to large alterations in electrolyte concentrations, deleteriously affecting neuronal excitability (Amiry-Moghaddam and Ottersen, 2003). Hence water transport across the BBB is intricately linked to ions transport. Brain perivascular astrocytic foot processes, sub-pial astrocytes and the ependyma, which lines the ventricles, express the water channels AQP-4 (Amiry-Moghaddam and Ottersen, 2003; Li et al., 2009; Nielsen et al., 1997). More recent studies demonstrated that brain endothelial cells also express AQP-4 at their luminal and abluminal membranes, albeit at a much lower density than in astrocytic foot processes (Amiry-Moghaddam and Ottersen, 2003; Kobayashi et al., 2001). The link between water movements and electrolyte concentrations is further strengthened by the co- expression of AQP4 and Kir-4.1 potassium channels, and the discovery that water move- ment is associated with potassium fluxes in the same direction (Amiry-Moghaddam and Ottersen, 2003; Niermann et al., 2001). The BBB is relatively permeable to water, allowing a slow bulk flow (Abbott, 2004) but less so to ions (Go, 1997). Electrolytes homeostasis in the ECF is actively maintained by the BBB, as evidenced by a high Na+, K+-ATPase activity in brain endothelial cells (Bauer et al., 1990; Go, 1997; Seda et al.,

1984). Potassium concentration in the ECF is kept at a lower level than that of plasma (Hansen, 1985). The blood-CSF barrier (BCSFB) The brain has four choroid plexuses that project into the two lateral ventricles and the third and fourth ventricles. Each choroid plexus contains a core of loosely arranged stroma derived from the leptomeninges. Their ventricular surface is extremely fringed (Fig.1). The core of the plexus is richly supplied with capillaries, the endothelial cells of which have fenestrations and leaky tight junctions (Wolburg and Paulus, 2010). Large molecules such as peroxidase penetrate the choroid plexus capillaries and enter the interstitial space in the core of the plexus (Brightman, 1968). As the plexus invaginates the ventricle it acquires a covering of ependyma, the cell layer that forms the lining of the ventricle (Fig.1). This ependymal covering is known as the choroidal epithelium, which, although continuous with the rest of the ependyma at the neck of the choroid plexus, is structurally and functionally different, as it becomes modified and specialized to form the BCSFB (Brightman 1968; Redzic and Segal 2004; Tripathi 1973). The choroidal epithelium therefore separates the CSF from the milieu of the plexus core with its leaky capillaries. Choroidal epithelial cells are joined by tight junctions, which occlude the paracellular route (Wolburg and Lippoldt, 2002; Wolburg and Paulus, 2010). Choroidal epithelial cells actively produce the CSF and regulate its electrolyte, protein and water content (Bradbury et al., 1963). Choroidal epithelial cells express the water channels AQP-1 (Agre et al., 1993; Nielsen et al., 1993). In addition to their main function of CSF secretion choroidal epithelial cells transport some substances from the blood to the CSF, for example, nucleotides and ascorbic acid, and actively removes other substances from the CSF (Go, 1997). There is no barrier between the brain and CSF located in the subarachnoid space, as the cells of the pia mater on the brain surface have gap junctions and no tight junctions (Alcolado et al., 1988). Peroxidase perfused into the subarachnoid space penetrates between pia mater cells, through the basement membrane of astrocytes at the brain surface and between astrocytes (outer glia limitans) to enter the neuropil 63

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(Brightman and Reese, 1969). Also free exchange occurs between the CSF located in the ventricles and the brain ECF across the ependymal lining of the ventricles, as peroxidase injected into the ventricles enter the neuropil (Brightman and Reese, 1969), thus the CSF and the ECF of the brain equilibrate. Magnesium transport across barrier membranes including the BBB and BCSFB Magnesium transport in the kidney The kidney is the main contributor to magnesium homeostasis, thus it is not surprising that research efforts to understand magnesium transport were focused on epithelial cells of the nephron. During the last decade greater understanding of the transport of magnesium across cell membranes was gained through genetic studies in families with hereditary abnormal magnesium balance, positional cloning and knockout mice investigations (Hoenderop and Bindels, 2005). Such studies led to the identification of genes encoding proteins involved in the transport of magnesium. One human gene, the paracellin-1 (PCLN-1), encodes the protein paracellin-1. PCLN-1 mRNA was detected using RT-PCR in the thick ascending limb of Henles loop and the distal convoluted tubule of the kidney in the rabbit (Simon et al., 1999) and rodents (Weber et al., 2001). The PCLN-1 protein was also detected using immunocytochemistry in the same segments in rabbit kidney using an antibody to PCLN-1 (Simon et al., 1999). Using confocal microscopy and double immuno- fluorescence for PCLN-1 and occuldin, a tight junction ubiquitous protein (Furuse et al., 1993), demonstrated colocalisation of the two proteins to intercellular tight junction at both segments of rabbit nephrons (Simon et al., 1999). The paracellin-1 protein is a member of the claudin family (claudin 16) (Simon et al., 1999). Families with mutation in PCLN-1 gene show hypo- magnesemia and renal magnesium wasting (Simon et al., 1999). The paracellin-1 located in the tight junction between epithelial cells in the nephron acts as a passive channel for reabsorbing magnesium along the paracellular route being favored by the positive luminal charge. Because these families also show hypercalciurea and urinary calculi, it was suggested that PCLN-1 is also a conductance for calcium (Simon et al., 1999). Such discovery modified the previously 64

prevalent view that tight junctions exist to prevent passage of molecules and are static; rather, they do provide selective barriers. Further studies on human recessive renal magnesium loss mapped a second locus on chromosome 1p34.2 and have identified mutations in CLDN19, which encodes the TJ protein claudin-19 expressed in the nephron tubules (Konrad et al., 2006). Recently plasma membrane cation channels that belong to the transient receptor potential (TRP) superfamily have been described and were classified into 7 subfamilies. The closely related subfamilies TRPC, TRPV, TRPM, TRPN and TRPA are classified as group 1, while group 2 includes TRPML and TRPP (Montell 2005). The TRPM subfamily includes 8 members, of which TRPM6 and TRMP7 have been recognized as highly permeable Mg2+ transporters into cells, and have been particularly studied in epithelial cells of the nephron (Aikawa 1976; Dai et al., 2001). In familial autosomal-recessive hypomagnesemia with sec-ondary hypocalcemia, affected infants suffer from seizures and tetany due to abnormal handling of Mg2+ in intestinal absorption and renal reabsorption. Genetic analysis pointed to a gene, TRPM6, which was mutated in these patients (Schlingmann et al., 2002; Walder et al., 2002). Using immunocytochemistry, it was shown that TRPM6 protein is localized to the apical membrane of renal distal convoluted tubules epithelium, the site of active reclaiming of magnesium, and also at the brush border of apical membranes of intestinal epithelium, the main site of magnesium absorption (Voets et al., 2004). Patch-clamp analysis and measurement of intracellular magnesium indicated that TRPM6 forms all or at least part of the magnesium channel in absorbing epithelia (Voets et al., 2004). It has been suggested that mammary epithelial cells in culture, with high and low magnesium content, adapt to low magnesium availability by upregulating magnesium influx via TRPM6, and to high magnesium availability by increasing magnesium efflux primarily via Na+/Mg2+ exchange (Wolf et al., 2010). The TRPM7 also has a central role in Mg2+ homeostasis, because TRPM7-deficient cells become Mg2+ deficient, and cannot survive (Schmitz et al., 2003). Magnesium transport across the BBB Magnesium entry into the brain correlates with

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maturation of the BBB. In fetal sheep and the guinea pig, there is a drop in the concentration of Mg2+ in the cerebral hemispheres, which corresponds approximately to the minimum in potassium content and the maximum in chloride and sodium contents (Bradbury et al., 1972). During rat postnatal development, brain content of magnesium shows regional variations. At day 5, magnesium is most marked in the pons and medulla and least marked in the cerebral cortex. Magnesium levels in all regions decline after day 5, in parallel with the decrease in water content and the increase in tissue weight, suggesting that the maturation of the BBB plays an important role in brain magnesium homeostasis (Chan et al., 1992). Magnesium is able to cross the BBB (Sacco et al., 2007), and it is transported via the barrier with a net flux from the blood into the parenchyma. Active magnesium transport from blood to the extracellular fluid of the brain is evidenced by its higher concentration in the cortical extracellular fluid than its concentration in the plasma- dialysate or cisternal CSF (Bito, 1969). Magnesium administration attenuates cell death due to cytoskeletal alteration (Saatman et al., 2001) and reduces apoptosis and the expression of p53 following TBI (Lee et al., 2004). Thus its administration rectifies the decline in intracellular Mg2+ levels and cellular functions (Saatman et al., 2001). In experimental closed head trauma, treatment with magnesium restores the polarity of astrocytes, in terms of aquaporin-4 distribution, to a preinjury state (Ghabriel et al., 2006). A similar protective effect for magnesium was demonstrated in induced hypoglycemia (Kaya et al., 2001). This indicates replenishing of magnesium in the extracellular space of the brain. However, the question remains, how does magnesium cross endothelial cells of the BBB? Is magnesium transport across the BBB similar to its transport across nephron epithelial cells? If so, does Mg2+ cross the BBB via a paracellular route or a transcellular route or both? Concerning the paracellular route, the proposed role for PCLN-1 (claudin-16) in the nephron (Simon et al., 1999), acting as a conductance for Mg2+, and its exclusive location in the kidney (Efrati et al., 2005; Weber et al., 2001), raise the possibility that other members of the claudin family may play a similar role for paracellular

conductance across other barrier membranes. Members of the claudin family are involved in the formation of TJ strands in various tissues (Morita et al., 1999). There is tissue-specific expression of claudin members in tight junctions, with a specific colocalisation of occludin with some but not all claudins (Peppi and Ghabriel, 2004). Magnesium is important for cell membrane stabilization. In the CNS, the myelin sheath is an elaboration of the cell membrane of oligodendrocytes, which express claudin-11. In claudin-11-deficient mice, the TJs are absent in the myelin sheath of oligodendrocytes and mice show demyelination (Gow et al., 1999). This indicates that claudin-11 is an important component in the stability and formation of the myelin tight junctions. Also endothelial cells of brain microvessels express several claudins, including claudin-1, -3, -5 and -12 (Coisne et al., 2005; Liebner et al., 2000; Matter and Balda, 2003; Morita et al., 1999; Tsukita and Furuse, 1999; Wolburg et al., 2003). Breakdown of the BBB in experimental autoimmune encephalo- myelitis in the mouse and the leaky vessels in human glioblastoma multiforme are accom- panied by selective loss of claudin-3 in BBB TJs (Wolburg et al., 2003). Although direct link between Mg2+ permeability and claudins has not been established in the CNS similar to the link between claudin-16 and epithelial cells of the nephron, it is tempting to speculate that, in addition to their supporting role in the morphology the BBB TJs, claudins in TJs of brain endothelial cells may contribute to Mg2+ conductance at the BBB. It is also speculated here that claudin-11 in the CNS myelin, in addition to its morphological supporting role, may act as a conductance for Mg2+ across the consecutive myelin lamellae, providing a faster access route from the ECF to the periaxonal space. Concerning the transport of Mg2+ across the BBB via a transcellular route, it may also be prudent to compare the BBB to other barrier membranes. As stated above, TRPM6 transporter protein has been localized to the apical membrane of renal distal convoluted tubules epithelium, the site of active reclaiming of magnesium (Voets et al., 2004), and TRPM7 in the kidney also has a central role in Mg2+ homeostasis (Schmitz et al., 2003). In mouse brain, using quantitative RT-PCR, TRPM3 and TRPM7 mRNA were detected at high levels, and 19 other isoforms were also present (Brown 65

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et al., 2008; Kunert-Keil et al., 2006). Also in human brain TRPM1, 2, 3, 6 and 7 have been detected using RT-PCR (Fonfria et al., 2006). Evidence of TRPM presence in brain endothelial cells was obtained from endothelial cell cultures. Immortalized mouse brain microvessel endo- thelial cells, freshly isolated cerebral microvessels and primary cultured rat brain endothelial cells express multiple TRPC and TRPV isoforms, and also TRPM2, M3, M4, and M7 mRNA (Brown et al., 2008). We would like to suggest therefore that the TRPM6 and TRPM7, the gatekeepers of magnesium (Schlingmann et al., 2007), are likely to play a main role in brain endothelial cell transport of magnesium via the transcellular route. Western blotting and immunofluorescence assays for TRPM6 and TRPM7 similar to those performed for TRPV4 in mouse cerebral microvascular endothelial cells (Ma et al., 2008) would support this suggestion. Also direct evidence may be obtained from measuring intracellular magnesium in endothelial cell culture similar to previous reports on mammary epithelial cells in culture (Voets et al., 2004) Magnesium transport across the blood-CSF barrier What has become apparent from experimental research and human studies is that magnesium concentration in the CSF is actively maintained above that of the plasma, and changes in CSF magnesium lag behind changes in its concentration in the plasma and are less pronounced (Bradbury and Sarna, 1977; Morris, 1992). Thus electrolyte composition in the CSF of adult mammals is different from that of plasma and is more stable, suggesting the existence of mechanisms for electrolyte homeostasis in the CSF, necessary for normal brain function (Bradbury et al., 1972; Somjen, 2002). The mechanisms determining the concentrations of ions in CSF appear to develop at different and largely independent rates (Bradbury et al., 1972). In the dog (Oppelt et al., 1963) and human (Nischwitz et al., 2008) the concentration of magnesium is higher in the CSF compared to plasma. Also in fetal sheep and guinea pigs magnesium concentration in the CSF is slightly higher than that of plasma (Amtorp and Sorensen, 1974; Bradbury et al., 1972). An investigation of the permeability of the human BCSFB to magnesium carried out in 29 individuals, using paired serum and CSF samples, showed a mean 66

CSF/serum ratio of 1.3 (Nischwitz et al., 2008). This study also compared magnesium to other metals, and suggested that low molecular weight species such as magnesium and calcium cross the BCSFB through less specific ion channels compared to high molecular weight metals such as iron, copper and zinc, which can pass the barrier only via well controlled receptor mediated pathways (Nischwitz et al., 2008) The choroidal epithelium plays an active role in maintaining the level of magnesium in the CSF by sensing changes in the CSF and altering the rate of active magnesium secretion (Oppelt et al., 1963; Reed and Yen 1978). Physiological studies of isolated choroid plexus of sheep (Allsop 1986) and cat (Reed and Yen 1978) showed that the choroid plexus is able to transfer magnesium against a concentration gradient. An in vivo study demonstrated a directional flow of magnesium into the CNS (Allsop and Pauli, 1985). In normal cows the magnesium concentration in the ventricular CSF was found to be higher than its concentration in the lumbar CSF (Allsop and Pauli, 1985). Under hypomagnesemic conditions, magnesium concentration in the ventricle decreased more rapidly than that of the lumbar CSF, while intravenous infusion of magnesium led to increased magnesium in the ventricular CSF before changes in the lumbar CSF (Allsop and Pauli, 1985). This study indicates that the access of magnesium from the blood to the brain is mainly via the choroid plexus to CSF, which equilibrates with the CNS parenchyma. Transport of magnesium in the choroid plexus is influenced by other selective electrolytes. For example, high levels of potassium in the perfusate of the choroid plexus leads to a reduction in the transfer rate of magnesium, but higher levels of calcium does not have a similar effect on the transfer of magnesium (Allsop, 1986). A recent clinical trial on the protective role of magnesium in traumatic brain injury (Temkin et al., 2007) failed to replicate the improved outcome detected in experimental studies on rodents (Temkin et al., 2007; Vink and Cernak, 2000). In this clinical trial, plasma magnesium was maintained at higher levels compared to the placebo group and normal plasma level, to counteract the reported decline in Mg2+ in TBI patients (Kahraman et al., 2003). Since under normal conditions choroidal epithelial cells

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actively transport magnesium into the CSF, it is likely that such activity was disrupted in TBI cases in humans leading and failure of magnesium entry to the CNS. It is relevant here to state that in the rat, TBI induces severe morphological changes in choroidal epithelial cells detected by scanning and transmission electron microscopy that were still evident 4 weeks after trauma (Ghabriel et al., 2010). Although one cannot dismiss the possibility that new channels may be involved in the transport of magnesium at the choroid plexus, it is natural to look at other systems for similar magnesium transport mechanisms. As stated above the strongest evidence for magnesium transport was obtained from genetic studies in familial hypomagnesemia, positional cloning and knockout mice investigations (Konrad et al., 2006; Schlingmann et al., 2005; Schlingmann et al., 2007; Simon et al., 1999; Walder et al., 2002) which identified TRPM6 and TRPM7 as candidates for transcellular transport, and Claudin16 (Efrati et al., 2005; Simon et al., 1999) for paracellular transport in the kidney. Recently TRPM7 has also been identified in smooth muscle cells in the vasculature (Callera et al., 2009).

Although TRPM6 and TRPM7 were detected in the brain using RT-PCR it is not clear if these channels are expressed in choroidal epithelial cells. The role of claudin-16 and claudin-19 in magnesium reabsorption from the kidney filtrate has been discussed in previous sections, and the role of claudins as selective conductance to ions through tight junction has been stated. The choroidal epithelial cells show selective expression of claudin-1, -2 and -5 at and near to their tight junctions. Also the endothelial cells within the choroid plexus show stronger expression of claudin-5 than claudin-1 and -2 (Lippoldt et al., 2000). However, claudin-16 and claudin-19 have not been reported in the choroid plexus. Whether other claudin members may contribute to magnesium conductance in the choroid plexus is speculative. Thus it appears that the channels for magnesium transport in the choroid plexus remain to be clarified. Acknowledgement Supported, in part, by funding from the Neurosurgical Research Foundation (Australia).


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Alcolado R, Weller RO, Parrish EP, Garrod D (1988) The cranial arachnoid and pia mater in man: anatomical and ultrastructural observations. Neuropathol Appl Neurobiol 14:1-17. Allsop TF (1986) Transfer of magnesium across the perfused choroid plexus of sheep. Aust J Biol Sci 39:161-9. Allsop TF, Pauli JV (1985) Magnesium concentrations in the ventricular and lumbar cerebrospinal fluid of hypomagnesaemic cows. Res Vet Sci 38:61-4. Altura BT, Altura BM (1991) Measurement of ionized magnesium in whole blood, plasma and serum with a new ion-selective electrode in healthy and diseased human subjects. Magnes Trace Elem 10:90-8. Amiry-Moghaddam M, Ottersen OP (2003) The molecular basis of water transport in the brain. Nat Rev Neurosci 4:991-1001.

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Intracellular free Mg2+ and MgATP2- in coordinate control of protein synthesis and cell proliferation
Harry Rubin*
Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA 94720-3200, USA.
* hrubin@berkeley.edu


Abstract Specific and non-specific mitogens stimulate the proliferation of cultured fibroblasts. They also stimulate other responses that are part of a coordinate response, some members of which are essential for proliferation and others that are not. The synthesis of protein is an early response to mitogens and its continuation through the G1 period drives the accumulation of protein which is required for the initiation of DNA synthesis hours later. The parabola-like curve for dependence of protein synthesis on intracellular Mg2+ concentration is similar to that of cell-free ribosome preparations, and is later reflected in the initiation of DNA synthesis. Hence, DNA synthesis is dependent on the rate of protein synthesis which is regulated by the concentration of intracellular Mg2+. Presumably free Mg2+ is an indicator of the fraction of ATP4- that is complexed with Mg2+ as MgATP2-, which is the immediate regulatory form. Uridine uptake is determined by its phosphorylation, which is also dependent on intracellular Mg2+ in the coordinate response, but unlike protein synthesis, neither influences DNA synthesis nor exhibits downturn at a high Mg2+ concentration. Intracellular free Mg2+ determines the onset of protein synthesis in activated frog oocytes and its rate in lymphocytes. Mg2+ regulation of protein synthesis is effected through the PI 3-K pathway at mTOR phosphorylation of two translation-regulating proteins. Regulation of proliferation by Mg2+ is lost in transformed cells. Mg2+ and MgATP2- apparently play a central role in the regulation of metabolism in a wide variety of cells and developmental stages across the animal kingdom.

Introduction The study of the regulation of cell proliferation was greatly facilitated in the 1960s and 1970s by the development of monolayer cell culture. This permitted direct microscopic examination of cell populations including cellular morphology and behaviour. Techniques were also developed for suspending and accurately counting the cells. It was also possible to vary the cell population size, change the constituents of the medium, and uniformly label the cells with radioactive chemicals to study their metabolism. Some general features of cell growth stimulation have to be considered before delving into the possible mechanism(s) of its control. Most of the experiments to be described here were conducted in cell culture with fibroblasts obtained directly from chicken embryos, or with established lines of mouse fibroblasts. In most cases they were allowed to grow into confluent sheets to establish contact inhibition with low levels of DNA synthesis that represented the small proportion of cells in the S-period, and no net

proliferation. Fresh medium containing serum or other growth stimulants was added and measure- ments of various cellular parameters were made at intervals thereafter. After application of a stimulus to a growth- inhibited culture, increase in DNA synthesis, representing the increased proportion of cells in the S-period, does not begin for 4 hours in ex vivo chicken embryo cells (Rubin and Steiner, 1975) or 10 hours in an established line of mouse cells (Rubin et al., 1978). This indicates that multiple metabolic processes are required to produce that response. These include increases in the rates of uptake of glucose analogues, and uridine prior to its incorporation into RNA (Rubin, 1975b). Although neither of these responses is required for the increased DNA synthesis, they are clearly part of a coordinate response of the cells to the growth stimulants, and have to be taken into account in understanding the intracellular mechanism of growth regulation. Most significant, there is also a fractional increase in the rate of protein synthesis that must be 75

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maintained throughout the period preceding DNA synthesis, and is essential for its initiation (Rubin, 2005). Small decreases of protein synthesis in cells treated with a specific inhibitor of protein synthesis result in large decreases in the fraction of cells that later initiate DNA synthesis (Brooks, 1977; and Figure 1). Another aspect of growth stimulation concerns treatments other than serum growth factors that produce the same constellation of responses. Certain hormones such as insulin produce the coordinate response in some cell types, indicating that a specific interaction between proteins and their receptors at the cell surface can initiate the coordinate response. However, the same effects are produced by removing a strip of cells from a confluent monolayer, which allows migration and multiplication of the cells at the margin. This indicates that the coordinate response is not limited to specific receptors. A similar conclusion is suggested by the finding that subtoxic concentrations of heavy metals including zinc, cadmium, and mercury initiate the coordinate response in chick embryo fibroblasts (Rubin, 1975b). Lead stimulated DNA synthesis in some experiments, and in other experiments did not (Sanui and Rubin, 1984). Those cultures in which DNA was not stimulated showed no increase in cellular Mg2+, whereas those that were significantly stimulated exhibited a 10 - 13% increase in intracellular Mg2+. Stimulation of DNA synthesis by lead coincided with the formation of lead precipitate in the medium. A striking finding made with a permanent line of cells originating from mouse embryo fibroblasts was that increasing concentrations of inorganic pyrophosphate initiated the coordinate response at precisely the concentration (about 0.2 mM in standard medium) at which it first formed a flocculent precipitate with calcium (Rubin and Sanui, 1977). The stimulation required contact of the precipitate with the cell surface as shown by the failure of stimulation when the culture dish was inverted so that the precipitate did not contact the cells (Bowen-Pope and Rubin, 1983). The stimulation was terminated when the pH of the medium was slightly lowered and the precipitate dissolved, showing that the effect required the precipitate and was exerted at the surface of the cell. 76

Figure 1. Inhibition of protein synthesis by cycloheximide and its effect on the initiation of DNA synthesis in mouse 3T3 cells. Quiescent cells were stimulated with serum in the presence of the indicated concentrations of cycloheximide. Cultures were pulse-labelled for protein synthesis with 3H-leucine at 2 hours () and continuously labelled with 3H-thymidine for 24 hours (O). Ordinate: radioactivity incorporated as percentage of control (no cycloheximide) (Brooks, 1977). In the absence of pyrophosphate, supranormal concentrations of either Ca2+ or orthophosphate caused graded increases in DNA synthesis (Rubin and Sanui, 1977). The effect of supranormal Ca2+ depended on orthophosphate and vice versa. The stimulation was associated with increasing turbidity of the medium, consistent with the involvement of a precipitate interacting with the cell membrane. Several other non-physiological, non-specific treatments could initiate the coordinate response, although most of them could not maintain multiple rounds of cell multiplication. One exception was the wound- healing experiment, which allowed continued cell multiplication until the cleared strip was completely covered with cells. The sum total of these experiments indicates that perturbation of the cell membrane, which could be brought about by specific or non-specific means, is involved in growth stimulation, and raises the question of what intracellular response(s) or

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second messenger(s) mediate the diverse reactions of the coordinate response. Variations of intracellular K+, Na+ or Ca+ failed to reproduce the coordinate response in a balanced manner (Moscatelli et al., 1979) nor did changes in the pH of the medium (Rubin, 2005). Intra- cellular Mg2+ was considered a logical candidate as a second messenger for regulating the coordinate response to membrane perturbation because it is required for every phosphoryl transfer in the cell. Drastic reduction in Mg2+ concentration in the medium reduced the number of cells in the S-period of the cell cycle, but the effect was erratic (Rubin, 1975a). Phosphorylated compounds that bind Mg2+ were more reproducible, especially inorganic pyro- phosphate in concentrations that exceeded the concentration of Mg2+ in the medium. Unlike the very low concentrations of pyrophosphate that stimulated mouse fibroblasts via a precipitate with Ca2+ (Bowen-Pope and Rubin, 1983), the inhibition of fibroblast growth was most efficient in low concentrations of Ca2+ in which there was no precipitate. Mg2+ deprivation, further lowered by its binding with pyrophosphate, suppressed all elements of the coordinate response in a balanced manner (Rubin, 1975a). Changes in total and free Mg2+ after stimulation of cells with mitogens The major cellular cations were measured in confluent chick embryo fibroblasts 16 hours after a fresh change of medium with insulin substituting for serum (Sanui and Rubin, 1978). There was more than a 16-fold increase in the overall rate of DNA synthesis in the optimal concentration of insulin, with a 22% increase in total Mg2+ of the cells as measured by atomic absorption spectro- photometry. There was no change in total cell Ca2+, negligible change in Na+ and a 14% increase in K+, an element that plays an adjunct role to Mg2+ in the synthesis of protein in cell-free systems (Schreier and Staehelin, 1973). In an experiment with a contact inhibited and serum- limited line of mouse fibroblasts, the addition of fresh medium containing serum induced a 15-fold increase of DNA synthesis at 17 hours, associated with a 15% increase in total Mg2+ (Sanui and Rubin, 1982). There was only a marginal increase in K+, and decreases in Na+ and Ca2+. These results

were consistent with a central role for Mg2+ in supporting cell proliferation. It seemed likely to us that intracellular free Mg2+ combined with ATP4- is its regulatory form, possibly made available by cation exchange with intracellular K+ that releases bound Mg2+ from the inner surface of the cell membrane (Rubin, 1976; Sanui and Rubin, 1978). The free Mg2+ would combine with ATP4- to form MgATP2-, which would stimulate phosphoryl transfer reactions throughout the cell, and reduce the inhibitory KATP3- form. However, we did not have an efficient means at the time of measuring free Mg2+ in the cell. The development of a Mg2+- sensitive fluorescent dye, mag-fura-2, made it possible to quantify intracellular free Mg2+ (Murphy et al., 1989; Raju et al., 1989). It was used to monitor free Mg2+ in a quiescent, confluent line of mouse fibroblasts stimulated by insulin or by insulin combined with epidermal growth factor (Ishijima et al., 1991). The combination of the two growth factors led to a significant increase of intracellular free Mg2+ from a basal level of 0.22 mM to 0.29 mM at 30 minutes, and to 0.35 mM at 60 minutes. After 60 minutes the free Mg2+ levels seemed to decline, but the measurements were no longer reliable because of leakage of the dye. Epidermal growth factor produced a 50-fold increase in DNA synthesis when added to a serum-starved line of differentiated myocytes (Grubbs, 1991). Free Mg2+ increased after a 5- minute lag period, rising gradually from an initial 0.32 mM to as high as 1.4 mM at 20 minutes, and then levelled off. There was no change in the cellular pH or free Ca2+ during the 20-minute period. The dose-response to epidermal growth factor was the same for free Mg2+ and the synthesis of DNA, strengthening the case for the dependence of the proliferative response on the rise of free Mg2+. Correlations between cellular Mg2+, protein synthesis and the onset of DNA synthesis A series of experiments was carried out in which external Mg2+ was varied over a wide range (1-48 mM) in a medium containing only 0.02 Ca2+ (Rubin et al 1979). The low Ca2+ concentration increased the permeability of the cells for entry of Mg2+. Intracellular Mg2+ content and the rate of protein synthesis were measured at 3 and 17 hours and the rate of DNA synthesis at 17 hours. 77

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The results showed that the intracellular Mg2+ concentration increased as the external Mg2+ concentration was increased, but the rate of protein synthesis at 3 hours peaked at 20 mM external Mg2+ concentration, and declined with higher (30 48 mM) Mg2+ concentrations (Figure 2A, B). The rate of DNA synthesis at 17 hours peaked at the same Mg2+ concentration as protein synthesis at 3 hours (Figure 2A), consistent with the dependence of the onset of the S-period on the rate of protein synthesis during the preceding G1-period.

Figure 2. Effects of extracellular Mg2+ on intracellular Mg2+ and on protein and DNA synthesis. Quiescent mouse 3T3 cells were stimulated with 10% dialyzed serum in Ca2+- deprived medium with varying concentrations of Mg2+. At 3 hours some of the cultures were A) labelled with 3H-leucine; and at 17 hours with 3H- leucine or 3H-thymidine; B) processed for measurement of intracellular Mg2+ at 3 and 17 hours (Rubin et al., 1979). Protein synthesis at 17 hours showed a stimulation at 30 mM external Mg2+ which had first inhibited protein synthesis at 3 hours, and consequently reduced DNA synthesis at 17 hours (Figure 2A). The increased protein synthesis at 17 hours was associated with a decrease of cellular Mg2+ from its inhibitory level at 3 hours to a physiological stimulatory level at 17 hours (Figure 2A, B), indicating the cells were able to reduce Mg2+ content to functional levels. If the external 78

Mg2+ levels were carried to even higher external levels (48 mM), the cellular Mg2+ rose to very high levels at 17 hours and both protein and DNA synthesis remained close to zero, indicating toxic damage of the cells. The overall results provided strong evidence of Mg2+ as an indirect regulator of the onset of DNA synthesis through its direct effect on the rate of preceding protein synthesis. It should be recalled however that the free Mg2+ level is an indicator of the fraction of ATP4- that is complexed to Mg2+ as MgATP2- which is the true regulator (Rubin, 2005). Figure 3 plots the rate of protein synthesis at 3 hours against the intracellular concentration of Mg2+, showing the sharp increase in protein synthesis with small increases of intracellular Mg2+ in the physiological range (0.05 0.08 mole/mg protein). At high concentrations of Mg2+, protein synthesis is reduced both in cultured cells and in cell free systems (Schreier and Staehelin, 1973; and Figure 4). The same concentration dependence is exhibited by DNA synthesis in cells at 17 hours (Rubin et al., 1978, and Figure 5), which is consistent with the dependence of DNA synthesis on prior protein synthesis. The increase of protein synthesis with ascending Mg2+ at physiological intracellular levels has to be maintained through the G-1 period in order to get the full response of DNA

Figure 3. Relationship between intracellular Mg2+ and protein synthesis. Quiescent mouse 3T3 cultures were stimulated with 10% dialyzed serum in Ca2+-deprived medium and varying concentrations of Mg2+. At 3 hours some of the cultures were labelled with 3H-leucine, or processed for measurement of intracellular Mg2+ (Rubin et al., 1979).

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Figure 4. Mg2+dependency of globin synthesis by mouse liver polysomes in a cell-free system, with labelling by 14C-leucine. After correction for chelation of Mg2+ by ATP and GTP, the effective Mg2+ concentration is 1.4 mM lower than that indicated (Schreier and Staehelin, 1973). The correction does not take into account that 32% of cytosolic Mg2+ is bound to low affinity, non- diffusible sites (Corkey et al., 1986). Note the steep increase in globin synthesis with Mg2+ at lower levels, closer to an estimated intracellular free Mg2+, if account is taken of all the low affinity binding sites. synthesis. The high concentration of total Mg2+ in cells, and the low affinity of Mg2+ binding sites, results in rather large changes of free Mg2+ with small variations in total cell Mg2+. This makes Mg2+ an unlikely short-term regulatory molecule like Ca2+, but long term change in total Mg2+, as exhibited in mitogen stimulation, may have profound effects on metabolic regulation given the sensitivity of free Mg2+ to alterations in total Mg2+ (Corkey et al., 1986). Response of protein synthesis in frog oocytes to injection of Mg2+ resembles that of stimulation by gonadotropin Frog oocytes are large enough (850-950 micrometres in diameter) to allow microinjection of materials, and to measure cations and protein synthesis in single cells (Horowitz and Lau, 1988). When stimulated by gonadotropin these quiescent cells begin to synthesize protein that drives their

Figure 5. DNA synthesis as a function of extra- cellular Mg2+. Quiescent mouse 3T3 cells were stimulated with 10% dialyzed serum in Ca2+- deprived medium with varying concentrations of Mg2+. At 17 hours the cultures were labelled with 3 H-thymidine. The asterisks associated with the two highest concentrations of Mg2+ in the medium and greatly reduced incorporation in DNA indicate many foci of degenerated cells (Rubin et al., 1978). growth and maturation into eggs. Exposing oocytes in saline to gonadotropin that acts via cell surface receptors increases cytoplasmic K+ activity and protein synthesis for at least 10 days (Lau et al., 1988). These effects are mimicked by microinjection of K+ into oocytes immersed in paraffin oil to prevent contributions from external sources. The results suggested that K+ activity is critical to activate and perpetuate protein synthesis. Further analysis of the results showed that there was a greater effect of K+ on translation in the oocytes than expected from its effect on cell-free translation (Horowitz and Tluczek, 1989). Also, injection of Na+ into the oocytes stimulated protein synthesis to the same extent as K+, despite the fact that Na+ has no effect on cell-free translation systems. These results indicated that Na+ and K+ compete with each other for binding to high affinity anions such as phosphate groups 79

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on membranes. Ca2+ was ruled out as the displaced cation, but Mg2+ stimulated protein synthesis at much lower and narrower concentrations than did K+. It was concluded that Mg2+, presumably as MgATP2-, is the effector directly controlling translation rates in oocytes. There was also evidence that stimulation of the oocytes by gonadotropin, which was necessarily done in saline medium containing Mg2+, added extracellular Mg2+ to that contributed by intracellular cation exchange (Horowitz and Tluczek, 1989). Role of Mg2+ in regulating uridine uptake The uptake rate of uridine into the acid soluble fraction of the cytoplasm increases several fold within a few minutes after stimulating cells with serum (Bowen-Pope and Rubin, 1977), and is sensitive to the concentration of Mg2+ in the medium (Rubin, 1976). External uridine is not required for growth of the cells, and its increased uptake does not require protein synthesis (Bowen-Pope and Rubin, 1977). The regulation of uridine uptake by Mg2+ occurs at its phosphorylation, which is the same step at which serum modulates uridine uptake (Vidair and Rubin, 1981). Phosphorylation of uridine is catalysed by uridine kinase, which responds to Mg2+ in cell-free systems in a manner similar to that exhibited by uridine uptake in living cells. Unlike protein synthesis, uridine uptake is not inhibited by high concentrations of Mg2+ in the cells, which is consistent with its simpler dependence on initial phosphorylation vs the many steps at which net protein synthesis may be affected by Mg2+ (Bowen-Pope et al., 1979). Thymidine uptake does not respond to mitogenic stimulation, nor does it require Mg2+ at the concentrations required by uridine (Vidair and Rubin, 1981). This is presumably because the Km of uridine kinase for Mg2+ is higher than that of thymidine kinase and within the normal range of intracellular Mg2+ variation (Vidair and Rubin, 2005). The divalent cation ionophore A23187 facilitates the manipulation of intracellular Mg2+ concentration without increasing the generalized permeability of the cell. In high concentrations of extracellular Mg2+, the ionophore-treated cells take up uridine in the absence of protein growth factors at the same high rate as in their presence. The results 80

support the thesis that increased uridine uptake in cells treated with growth factors is determined by a membrane-activated increase in intracellular free Mg2+. Molecular studies of Mg2+ regulation in lymphocytes TRPM7 is a membrane protein with intrinsic ion channel and protein kinase domains (Schmitz et al., 2003). An established line of chicken lymphocytes deficient in TRPM7 entered a quiescent phase when cultured in standard concentrations (0.4 mM) of Mg2+ similar to that of lymphocytes in the organism. Raising the Mg2+ concentration in the medium to 15 mM resulted in an increase in both the viability and proliferation of the cells, implying increased protein accumulation accompanying progress through the cell cycle. This activation indicated that TRPM7 has an essential role in regulating physiological concentrations of Mg2+ by an uptake pathway with functional coupling between its channel and kinase domains. TRPM7 channels are required for PI 3-K function as a central regulator of growth in lymphocytes (Sahni and Scharenberg, 2008), as had been hypothesized from studies of Mg2+ regulation in fibroblasts (Rubin, 2005). The PI 3-K pathway leads through a protein kinase cascade to mTOR kinase which is a central regulator of protein synthesis (Schmelzle and Hall, 2000). mTOR accomplishes this role by phosphorylating serines and threonines on two proteins which drive further steps that initiate translation (Terada et al., 1994; Sahni and Scharenberg, 2008). The mTOR phosphorylations have a high Km for MgATP2-, which is in the physiological range of free Mg2+. Total Mg2+ in the TRPM7-deficient lymphocytes in standard medium underwent a significant decrease, but no change in free Mg2+ was detected, which contrasts with the increase in free Mg2+ in mitogen-stimulated fibroblasts (Ishijima et al., 1991) and in myocytes (Grubbs, 1991). A number of the known properties of quiescent, ex-vivo lymphocytes were also measured in the TRPM7-deficient lymphocytes in standard medium containing 0.4 mM Mg2+ (Sahni et al., 2010). The DNA content and size of the two cell sources were similar, and suggested that TRPM7 and Mg2+ are involved in the activation of normal quiescent lymphocytes. The TRPM7-deficient

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lymphocytes increased in size and entered rapid proliferation when the concentration of Mg2+ in the medium was increased. The key cell regulator p27 was elevated in both normal, quiescent lymphocytes and in TRPM7-deficient cells in standard medium. A reduction in RNA, store- operated Ca2+ entry and altered energy metabolism was found in the quiescent TRPM7- deficient cells and in normal quiescent lymphocytes. The addition of excess Mg2+ to the deficient cells altered all these processes in a characteristic manner of activation of normal lymphocytes, just as it activated the coordinate response of fibroblasts in Ca2+-deficient medium (Rubin et al., 1979). The implication of these and foregoing results is that cytosolic free Mg2+ and MgATP2- play a central role in the regulation of all cell types and stages of development across the animal kingdom. Loss of Mg2+ regulation in neoplastically transformed cells Neoplastically transformed cells have the capacity to proliferate in lower concentrations of serum and to higher saturation densities than non-transformed cells (Rubin, 1981). Cell transformation causes a selective loss of the regulatory role of Mg2+ in cellular multiplication (McKeehan and Ham, 1978). Deprivation of Mg2+ caused transformed cells to flatten and to assume a regularly patterned, non-overlapping relationship to one another (Rubin et al., 1981). After 3 days of Mg2+ deprivation, the rate of DNA synthesis became highly dependent on both serum concentration and population density, thus resembling the growth behaviour of non- transformed cells. The capacity of the normalized transformed cells to produce colonies in agar in normal Mg2+ is greatly reduced after 7 days of Mg2+ deprivation in monolayer culture (Rubin and Chu, 1982). The Ca2+ content of transformed cells is only one- third that of non-transformed cells, but it increases to that of the non-transformed cells in Mg2+-deprived medium (Rubin et al., 1981). The normalization of these parameters in Mg2+- deprived transformed cells is reversed a few days after their subculture in Mg2+-adequate medium. The results support the suggestion that a defect in Mg2+ regulation of the multiplication of transformed cells is a basic feature of neoplastic

transformation (McKeehan and Ham, 1978) which could be exerted at the level of protein synthesis (Rubin, 2005; Sahni and Scharenberg, 2008). Nothing has been established about the cause of the postulated loss of Mg2+ control in transformed cells but one possibility has to do with their actively moving surface membranes (Abercrombie and Ambrose, 1958), which could result in lowering of the binding of Mg2+ to anionic groups on their internal surface, and its release into the cytosol (Rubin, 2005). This, of course, would shift the question to what causes the excessive membrane activity, which gets into the somatic cell genetics of neoplastic transformation, and is beyond the scope of this article. Conclusions There were several lines of thought that led to the suspicion that Mg2+ plays a central role in regulation of growth and replication. Among them were the observations that a variety of unrelated and non-specific substances could initiate at least one round of DNA synthesis and cell replication (Rubin, 1975b; Rubin and Sanui, 1977). Another was that a number of reactions were activated by mitogenic substances that were essential for cell replication and others that were not, but all responded directly to the mitogens hours before the synthesis of DNA was initiated (Bowen-Pope and Rubin, 1977). These early responses were considered part of a coordinate response and drove a search for a second messenger that could regulate all members of that response. That search was pursued by depriving confluent, contact-inhibited cultures of each extracellular candidate in turn, and determining the capacity of its removal to inhibit both the early responses and later increases in DNA synthesis of freshly stimulated cultures (Rubin, 1976; Rubin et al., 1979). The most significant early response of the cells for controlling later DNA synthesis was the synthesis of proteins, and the only measured cation that correlated quantitatively with both reactions was total intracellular Mg2+ concentration produced by variation in extracellular Mg2+. In order to reliably produce a change of intracellular Mg2+ over a wide range, it was necessary to drastically lower the concentration of Ca2+ in the medium while varying the extracellular concentration of Mg2+. The deprivation of Ca2+ increased the 81

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permeability of the cells for Mg2+ as well as for other small molecules. It was concluded that the concentration of free Mg2+ in the cells determined the level of the coordinate response, and most significantly of protein synthesis. This conclusion was in part based on small increases in total Mg2+ assuming that there were accompanying increases in free Mg2+. Supporting evidence for this conclusion was provided by experiments in Xenopus oocytes which allowed direct injection and measurement of free Mg2+ (Horowitz and Tluczek, 1989). However, there was a failure to demonstrate an increase in free Mg2+ by indicator measurements in activated lymphocytes (Rink et al., 1982), despite the reported increase in total Mg2+ (Sahni et al., 2010). It should be noted that although Mg2+ is a co-factor in all phosphoryl transfers in cells, the actual regulatory substrate is MgATP2- in which the Mg2+ is chelated by ATP4-, and therefore would not be registered by indicators of free Mg2+. The PI 3-K pathway is essential for the regulation of protein synthesis (Richardson et al., 2004). It leads through a protein kinase cascade to mTOR kinase, which is considered a central regulator of protein synthesis (Schmelzle and Hall, 2000). mTOR accomplishes this role by phosphorylating serines and threonines on two protein substrates which regulate the initiation of translation, mainly of ribosomal proteins and elongation factors (Terada et al., 1994). The mTOR phosphorylations have a high Km for MgATP2- (Dennis et al., 2001; Jaeschke et al., 2004). It may be significant for the coordination of protein synthesis with energy production that the kinases References
Abercrombie M, Ambrose EJ (1958) Interference microscope studies of cell contacts in tissue culture. Exp Cell Res 15:332-45. Bowen-Pope DF, Rubin H (1977) Magnesium and calcium effects on uptake of hexoses and uridine by chick embryo fibroblasts. Proc Natl Acad Sci USA 74:1585-9.

of carbohydrate metabolism which phosphorylate low molecular substrates have a Km for MgATP2- similar to that of mTOR (Edelman et al., 1987; Garner and Rosett, 1973). Hence MgATP2- may have as important a role in regulating energy production as it does in regulating protein and DNA synthesis. One aspect of Mg2+ regulation that needs more investigation is the putative role of the loss of its function in cell transformation (McKeehan and Ham, 1978; Rubin et al., 1981). A major target for mTOR phosphorylation is the eukaryotic initiation factor 4E (eIF4E). Enhanced phosphorylation of eIF4E up-regulates several proteins implicated in tumorigenesis (Furic et al., 2010). Since mTOR is itself regulated by MgATP2- (Rubin, 2005; Sahni and Scharenberg, 2008), the loss of regulation by Mg2+ in tumorigenesis, if confirmed, should lead to increased activity of eIF4E and could account for its frequent involvement in human cancer (Hay, 2010). Hence, the measurement of free Mg2+ in neoplastic transformation would be a logical next step. Acknowledgements I am grateful to Dorothy M. Rubin for editing the manuscript. The meticulous preparation of material and measurements of cations by the late Dr. Hisashi Sanui in cells and media by atomic absorption spectrophometry lent great accuracy and reliability to the results. The work of graduate students Dan Bowen-Pope, Dave Moscatelli, Mark Terasaki and Charlie Vidair contributed much in their day to an under- standing of the role of Mg2+ in the coordinate response of cells to mitogenic stimuli.


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McKeehan W, Ham RG (1978) Calcium and magnesium ions and the regulation of multiplication in normal and transformed cells. Nature 275:756-8. Moscatelli D, Sanui H, Rubin H (1979) Effects of + + 2+ depletion of K , Na , or Ca , on DNA synthesis and cell cation content in chick embryo fibroblasts. J Cell Physiol 101:117-28. Murphy E, Freudenrich CC, Levy LA, London RE (1989) Monitoring cytosolic free magnesium in cultured chicken heart cells by use of the fluorescent indicator Furaptra. Proc Natl Acad Sci USA 86:2981-4. Raju B, Murphy E, Levy LA, Hall RD, London RE (1989) A fluorescent indicator for measuring cytosolic free magnesium. Am J Physiol 256:C540-C8. Richardson CJ, Schalm SS, Blenis J (2004) PI3-kinase and TOR: PIKTORing cell growth. Seminars in Dev Biol 15:147-59. Rink TJ, Tsien RY, Pozzan T (1982) Cytoplasmic pH and 2+ free Mg in lymphocytes. J Cell Biol 95:189-96. Rubin H (1975a) Central role for magnesium in coordinate control of metabolism and growth in animal cells. Proc Natl Acad Sci USA 72:3551-5. Rubin H (1975b) Nonspecific nature of the stimulus to DNA synthesis in cultures of chick embryo cell. Proc Natl Acad Sci USA 72:1676-80. Rubin H, Steiner R (1975) Reversible alteration in the mitotic cycle of chick embryo cells in various states of growth regulation. J Cell Physiol 85:261-70. Rubin H (1976) Magnesium deprivation reproduces the coordinate effects of serum removal or cortisol addition on transport and metabolism in chick embryo fibroblasts. J Cell Physiol 89:613-26. Rubin H, Sanui H (1977) Complexes of inorganic pyrophosphate, orthophosphate and calcium as stimulants of 3T3 cells multiplication. Proc Natl Acad Sci USA 74:5025-30. Rubin H, Terasaki M, Sanui H (1978) Magnesium reverses inhibitory effects of calcium deprivation on coordinate response of 3T3 cells to serum. Proc Natl Acad Sci USA 75:4379-83. Rubin H, Terasaki M, Sanui H (1979) Major intracellular cations and growth control: Correspondence among magnesium content, protein synthesis, and the onset of DNA synthesis in Balb/c 3T3 cells. Proc Natl Acad Sci USA 76:3917-21.

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Rubin H (1981) Growth regulation, reverse transformation, and adaptability of 3T3 cells in 2+ decreased Mg concentration. Proc Natl Acad Sci USA 78:328-32. Rubin H, Vidair C, Sanui H (1981) Restoration of normal appearance, growth behavior and calcium content to transformed 3T3 cells by magnesium deprivation. Proc Natl Acad Sci USA 78:2350-4. Rubin H, Chu B (1982) Self-normalization of highly transformed 3T3 cells through maximized contact interaction. Proc Natl Acad Sci USA 79:1903-7. 2+ 2- Rubin H (2005) Central roles of Mg and of MgATP in the regulation of protein synthesis and cell proliferation: significance for neoplastic transformation. Adv Cancer Res 93:1-58. Sahni J, Scharenberg AM (2008) TRPM7 ion channels are required for sustained phosphoinositide 3-kinase signaling in lymphocytes. Cell Metabolism 8:84-93. Sahni J, Tamura R, Sweet I, Scharenberg AM (2010) TRPM7 regulates quiescent/proliferative metabolic transitions in lymphocytes. Cell Cycle 9:3565-74. Sanui H, Rubin H (1978) Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells. J Cell Physiol 96:265-78.

Sanui H, Rubin H (1982) Changes of intracellular and externally bound cations accompanying serum stimulation of mouse Balb/c3T3 cells. Exp Cell Res 139:15-25. Sanui H, Rubin H (1984) Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells. Biological Trace Element Research 6:289-307. Schmelzle T, Hall MN (2000) TOR, a central controller of cell growth. Cell 103:253-62. Schmitz C, Perraud A-L, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg A 2+ (2003) Regulation of vertebrate cellular Mg homeostasis by TRPM7. Cell 114:191-200. Schreier MH, Staehelin T (1973) Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems. J Mol Biol 73:329-49. Terada N, Patel HR, Takase K, Kohno K, Nairns AC (1994) Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci USA 91:11477-81. 2+ Vidair C, Rubin H (1981) Evaluation of Mg as an intracellular regulator of uridine uptake. J Cell Physiol 108:317-25. 2+ Vidair C, Rubin H (2005) Mg as activator of uridine phosphorylation and other cellular responses to growth factors. Proc Natl Acad Sci USA 102:662-6.

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Magnesium and the Yin-Yang interplay in apoptosis


Valentina Trapani, Lucia Mastrototaro and Federica I. Wolf *
Istituto di Patologia Generale e Centro di Ricerche Oncologiche Giovanni XXIII, Universit Cattolica del Sacro Cuore, Rome, Italy.
* fwolf@rm.unicatt.it

Abstract Apoptosis is a distinctive feature in the physiology of the developing brain, but also a key event in pathological conditions of the adult brain. The basic mechanisms executing cell death by apoptosis are conserved among different tissues and in different conditions, while the initiating event(s) may be more specific. Magnesium appears to be an important player in the process, though it might exert opposite actions depending on extra/intracellular availability. Extracellular magnesium deficiency induces apoptosis, mainly through increased oxidative stress, while intracellular magnesium mobilization from intracellular stores and consequent increase of cytosolic free magnesium seem to act in the effector phase. The molecular mechanism and the physio-pathological meaning of these findings await further characterization. The issue is even more complex in the context of the brain, where many concurring factors may determine a pro- or anti-apoptotic environment. A deeper understanding of the yin-yang role of magnesium in apoptosis may cast light on the basic processes that regulate cell fate, and consequently may open up novel opportunities for a successful therapeutic intervention for all the pathological conditions where excessive and undue apoptosis takes place.

Cell death by apoptosis Multicellular organisms maintain their homeo- stasis thanks to a tightly regulated mode of cell death. Programmed cell death, or apoptosis, is an internally controlled suicide program consisting in a stereotyped sequence of biochemical and morphological changes that allow the cell to die without adversely affecting its neighbours, i.e. without causing inflammation. As such, apoptosis is distinct from necrosis, the traumatic form of cell death whereby massive disruption of the surrounding tissue takes place. A cell may be doomed to die either by apoptosis or necrosis depending on the intensity and duration of the stimulus, the rapidity of the death process, and the extent of ATP depletion suffered by the cell. Apoptosis occurs in numerous physiological, adaptive and pathological events: 1) during development; 2) as a homeostatic mechanism to maintain cell populations in proliferating tissues; 3) as a defence mechanism such as in immune reactions; 4) following cell injury from various agents; and 5) in aging. There is general agreement that this form of cell death is an energy-dependent cascade of molecular events leading to cell shrinkage with maintenance of organelle integrity; long-recognized features of

apoptosis are protein cleavage and cross-linking, chromatin condensation, DNA fragmentation and, ultimately, phagocytic recognition and engulfment of the dying cell. The process of apoptosis is triggered by a diverse range of cell signals, which may originate either extracellularly (i.e. death receptor ligation with their cognate ligands, extrinsic pathway) or intracellularly (e.g. DNA damage or organelle dysfunction, intrinsic pathway). The apoptotic cascade consists of three parts: first, cells process the receptor-mediated death signal (initiation phase, somewhat more cell- specific), then get ready to implement apoptosis (effector phase, more conserved among different tissues) and finally commit suicide (degradation phase). During the initiation phase, the receptor- mediated death signal activates an intracellular cascade of events including: 1) activation of initiator caspases (caspase-8, for example), that can act early in the cell death process before, or independently of, mitochondrial changes; 2) increase in levels of oxyradicals and Ca2+; 3) transcription and translocation of pro-apoptotic Bcl-2 family members (Bax and Bad) to the mitochondrial membrane. The execution phase involves: 1) increased mitochondrial Ca2+ and oxyradical levels; 2) opening of permeability 85

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transition pores (PTP) in the mitochondrial membrane; 3) release of cytochrome c and many other apoptosis-inducing factors from the mitochondrial matrix into the cytosol; 4) formation of a molecular complex (the apopto- some) between cytochrome c and apoptotic protease-activating factor 1 (Apaf-1), that recruits and activates pro-caspase-9. Activated caspase-9, in turn, activates caspase-3, which begins the degradation phase: proteins are cleaved by various caspases and packed by trans- glutaminases, while nucleic acids are cleaved by activated Ca2+/Mg2+ endonucleases, which results in the formation of apoptotic bodies and subsequent phagocytosis by elicited macro- phages or neighbouring cells. It is interesting to underline the role of mitochondria in triggering apoptosis. Whether or not a direct injury jeopardizing the efficiency of these energy producing organelles occurs, mitochondria play a pivotal role in the cell death decision, as apoptotic factors are released by active mechanisms, which can be unrelated to mitochondrial damage (Tait and Green, 2010). Role of Magnesium in Apoptosis In the attempt to review the role of magnesium in apoptosis, one should first of all keep in mind that the effects of extracellular magnesium availability may differ from those due to intracellular magnesium fluxes or mobilization. In fact, decreased extracellular magnesium availability does not necessarily reflect an intracellular depletion, as demonstrated by the fact that in vivo serum magnesium levels are not a reliable marker of pathological hypo- magnesemia. Sensitivity to extracellular magnesium availability is highly tissue-specific, and can be translated in molecular terms by considering the expression and activity of magnesium-specific cation channels, such as the transient receptor potential melastatin (TRPM) -6 and -7 channels, which, by modulating trans- membrane cation fluxes, regulate the intracellular ion concentration (Schlingmann et al., 2007). Furthermore, intracellular magnesium is tightly buffered to meet specific metabolic requirements. As discussed elsewhere in this book, large changes in total cell magnesium occur with little or no change in cytosolic free magnesium, suggesting that the changes in total magnesium are due to changes in bound or 86

sequestered magnesium (Murphy, 2000). ATP is the most important intracellular magnesium buffer. Following massive ATP hydrolysis intra- cellular free magnesium transiently increases and is subsequently released into the extracellular environment. As a consequence, in a dying cell an increase in ionized magnesium occurs. Therefore, caution should be used also when investigating the role of intracellular magnesium as a player in the apoptotic program, in order to discriminate coincidental from causative events. In conclusion, whether considering extracellular magnesium availability or intracellular magnesium concentration, the story is more complicated than it appears at first glance. We will attempt to discuss the issue by reviewing the latest available data in view of the complex cellular physiology of magnesium. Extracellular magnesium and apoptosis Several important aspects of magnesium biochemistry and physiology point to a possible role for this cation in the apoptotic process. As discussed in other chapters of this book, magnesium is a key modulator of cell proliferation and metabolism and, most importantly, magnesium availability appears to affect the occurrence of oxidative stress. Many studies are available in the literature on the subject, but the most convincing ones investigate the effects of hypomagnesemia in vivo. In these circumstances, however, other factors, including inflammation, cytokine production and activation of phagocyte oxidative burst, concur to what is referred to as the pro- oxidant effect of hypomagnesemia (Mazur et al., 2007). Induction of apoptosis mediated by oxidative stress following magnesium deprivation has been documented in several tissues. Accelerated thymus involution, a classical example of apoptosis, was found in magnesium- deficient rats (Malpuech-Brugre et al., 1999). Dietary magnesium deficiency also induced apoptosis in cardiovascular tissues (Altura et al., 2009; Tejero-Taldo et al., 2007) and in the liver (Martin et al., 2003; Martin et al., 2008). In addition to the induction of reactive oxygen species, low magnesium availability could in principle trigger apoptosis by affecting DNA structure. This occurs not only by promoting oxidative DNA damage, but also by impairing DNA repair mechanisms, since magnesium is required for several crucial DNA repair enzyme

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activities, e.g. endonucleases, ligases, topo- isomerases (Hartwig, 2001). Intracellular magnesium and apoptosis As to intracellular events, several in vitro studies have suggested a promoting role for magnesium in apoptosis. An early increase in intracellular magnesium seems to follow both extrinsic and intrinsic induction of apoptosis (Patel et al., 1994; Chien et al., 1999; Zhang et al., 2005). The most straightforward explanation is that the increase in intracellular magnesium concentration is necessary for stimulating the activity of Ca2+/ Mg2+dependent endonucleases, which perform the apoptotic event par excellence, i.e. nucleosomal DNA fragmentation. Interestingly, however, the source of intracellular magnesium was hypothesized to be in the mitochondria (Chien et al., 1999), which is particularly appealing, as it is well established that mitochondria play a central role in the onset of the apoptotic program. Other lines of evidence point to the same direction. While the opening of the permeability transition pores appears to be dispensable for the release of cytochrome c, this is not the case for the presence of magnesium, which instead seems an absolute requirement (Eskes et al., 1998; Kim et al., 2000). Another study (Salvi et al., 2004) indicated that an apoptotic compound, gliotoxin, can specifically activate a magnesium efflux system from mitochondria in conditions of preserved mitochondrial integrity (i.e. high membrane potential, no swelling and retention of other ions). Most importantly, it has been shown that mitochondria a ct i n f act a s m agnesium s tores, Table 1: Magnesium and apoptosis EXTRACELLULAR DECREASED MAGNESIUM Impaired trophic signalling factor Increased oxidative stress Increased DNA damage Decreased DNA repair

and that Mg2+ release can occur following Ca2+ release and prior to ATP hydrolysis (Kubota et al., 2005). It is noteworthy that the same Authors have recently demonstrated that glutamate administration to rat hippocampal neurons triggers the same pathway, whereby Ca2+ accumulation in the mitochondria is required for Mg2+ release from the organelles (Shindo et al., 2010). The presence of a specific mitochondrial channel for magnesium, Mrs2, (Kolisek et al., 2003) corroborates these findings. As the activity of Mrs2 is dependent from membrane potential, it can be speculated that stored magnesium might be released through the channel upon depolarization. Intriguingly, in a recent paper Mrs2 expression has been associated with resistance to drug-induced apoptosis in cancer cells (Chen et al., 2009): by upregulating magnesium uptake into mitochondria, Mrs2 might counteract the increase in cytosolic magnesium that seems to be necessary for the execution of the apoptotic program via the mitochondrial pathway (Wolf and Trapani, 2009). In conclusion, the latest findings seem to concur to suggest a finer involvement of magnesium in the apoptotic cascade: not just a biochemical factor, but rather a crucial control element in life vs death decisions (see Table 1 for a summary). At present, these are only attractive speculations; most importantly, it has to be clarified whether the increase in intracellular magnesium occurring following an apoptotic stimulus is just a coin- cidental event (for example, due to mitochondrial depolarization) or rather a causative determinant in the downstream signalling cascade.

INTRACELLULAR INCREASED MAGNESIUM Mitochondrial damage Mitochondrial dysfunction Ca2+ signals Excitotoxicity


EXTRINSIC AND INTRINSIC PATHWAY

INTRINSIC PATHWAY
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Neuronal Apoptosis In contrast to the rapid turnover of cells in proliferative/renewing tissues, neurons commonly survive for the entire lifetime of the organism this enduring nature of neurons is necessary for maintaining the function of those cells within neuronal circuits. However, during development of the central and peripheral nervous systems, many neurons undergo apoptosis during a time window that coincides with the process of synaptogenesis (Oppenheim, 1991). Initial overproduction of neurons, followed by death of some, is probably an adaptive process that provides enough neurons to form nerve cell circuits that are precisely matched to their functional specifications. However, the persistence of neurons throughout life to preserve brain function implies that a considerable evolutionary pressure was placed on the development of mechanisms that guarded against neuronal death and/or promoted neuronal survival and plasticity. Trophic factors Like all cells, neuronal survival requires trophic support. Viktor Hamburger and Rita Levi- Montalcini described in a seminal paper that the survival of developing neurons is directly related to the availability of their innervating targets (Hamburger and Levi-Montalcini, 1949). This laid the foundation for the neurotrophin hypothesis, which proposed that immature neurons compete for target-derived trophic factors that are in limited supply. Neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4/5 (Lewin and Barde, 1996), generally activate and ligate the Trk receptors (TrkA, TrkB and TrkC), which are cell-surface receptors with intrinsic tyrosine kinase activity. They can autophosphorylate; for instance, after the binding of NGF to TrkA, the receptor phosphorylates several tyrosine residues within its own cytoplasmic tail. These phosphotyrosines in turn serve as docking sites for other molecules such as phospholipase Cg, phosphoinositide 3- kinase (PI(3)K) and adaptor proteins such as Shc, and these signal transduction molecules coordinate neuronal survival through the Akt and MAP kinase signalling pathways (Hennigan et al., 2007). Several neurotrophic factors and cytokines use a survival pathway involving the transcription factor NF-B (Mattson and Camandola, 2000). 88

The neurotrophin hypothesis predicts correctly that neuronal survival requires a positive survival signal. It does not, however, provide a concrete hypothesis as to how neurons die in the absence of trophic support; it was assumed that neurons die simply of passive starvation. In 1988, using cultured sympathetic neurons as a model system, Johnson and colleagues showed that inhibition of RNA and protein synthesis blocked sympathetic neuronal cell death induced by nerve growth factor (NGF) deprivation (Martin et al., 1988), providing the first tangible evidence that neurons might actually actively instigate their own demise. Glutamate receptors Lack of neurotrophic support is undeniably the best-studied signal that may trigger apoptosis during development of the nervous system. However, most neurons in the mammalian central nervous system possess receptors for another trigger of apoptosis, glutamate. Glutamate is a major excitatory neurotransmitter with a crucial role in neural development, synaptic plasticity, and learning and memory under physiological conditions (Riedel et al., 2003). Glutamate receptors are classified into several classes. In this context, we will go into some detail only for N-methyl-D-aspartate (NMDA) receptors. NMDA receptors are tetra- heteromeric ligand-gated ion channels that open upon the binding of glutamate. Magnesium binds the channel pore in a voltage-dependent manner; thus, at normal physiological resting membrane potential, the NMDA receptor is blocked by Mg2+. Synaptic release of glutamate causes Na+ influx through -amino-3-hydroxy-5-methyl-4-isoxazole- propionate (AMPA) receptors in the postsynaptic cell, resulting in partial membrane depolarization sufficient to lift the Mg2+ block and activate the channel. The activated NMDA receptor is permeable to Na+ but, crucially, also to Ca2+. This Ca2+ influx mediates most of the physiological effects of NMDA receptor activity, leading to postsynaptic depolarization and action potential in the postsynaptic neuron. Physiological levels of synaptic NMDA receptor activity are essential for neuronal survival (Hetman and Kharebava, 2006). However, regulation of glutamatergic neurotransmission is critical, as improper management of glutamate levels and glutamate receptor activity may impair

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not only its signalling properties, but can lead to cell death. The concept of excitotoxicity was first proposed by John Olney in 1969 as a toxic effect of excessive or prolonged activation of receptors by excitatory amino acids (Olney, 1969). Although the molecular pathways involved in excitotoxicity are still not fully understood at the present, there is a wealth of evidence suggesting that over- stimulation of glutamate receptors produces multiple adverse effects including impairment of intracellular calcium homeostasis, dysfunction of mitochondria and endoplasmic reticulum, increase in nitric oxide (NO) production and free radicals, persistent activation of proteases and kinases, increases in expression of pro-death transcription factors and immediate early genes (IEGs), ultimately leading to apoptosis (Wang and Qin, 2010). Excitotoxicity might mediate neuronal damage in various neurological disorders including ischemia and traumatic brain injury (Arundine and Tymianski, 2004) and neurodegen- erative diseases (Lipton and Rosenberg, 1994; Rego and Oliveira, 2003); it has also been implicated in neonatal brain injury (Johnston, 2005). NMDA receptors are important mediators of glutamate- induced excitotoxicity, as calcium entering through over-activated NMDA receptors results in more cell death as opposed to calcium entering through non-NMDA glutamate receptors or voltage-gated calcium channels (Cristofanilli and Akopian, 2006), which maybe due in part to their high permeability to Ca2+ and incomplete desensitization. Thus, responses to NMDA receptor activity follow a classical hormetic dose-response curve: both too much and too little can be harmful (Hardingham, 2009). Pathological stimuli Another trigger of neuronal death is increased oxidative stress, whereby free radicals (such as the superoxide anion radical and the hydroxyl radical) damage cellular lipids, proteins and nucleic acids by disrupting chemical bonds in those molecules. Metabolic stress, in which levels of glucose, oxygen and other molecules required for ATP (energy) production are decreased, and environmental toxins may also initiate neuronal apoptosis (Mattson, 2000). These stimuli are mostly involved in aging and pathological conditions, such as acute or chronic neuro- degenerative diseases. The classical and most

studied pathway of oxidation-induced apoptosis consists in cell-damage triggered phosphorylation of p53 and transcription of pro-apoptotic factors like Bid or Bad leading to mitochondrial- dependent activation of caspase 9. The genetic and environmental factors that trigger neuronal apoptosis may be different in various physiological and pathological settings and can vary from those active in other tissues, but most of the subsequent biochemical events that execute the cell death process are highly conserved and shared with all other cell types. Thus, the key components of the apoptosis program in neurons, like that of other cell types, are Apaf-1 and proteins in the Bcl-2 and caspase families. Nevertheless, different types of neurons, and neurons at different developmental stages, express different combinations of Bcl-2 and caspase family members, which is one way of providing the specificity of regulation (Yuan and Yankner, 2000). Tissue specific factors An alternative way to control cell death or survival is through the expression of tissue- specific proteins that affect signal transduction reactions and may be either pro-apoptotic or pro- survival. The prostate apoptosis response-4 (Par-4) protein was identified as being upregulated in prostate tumour cells undergoing apoptosis, but is now known to be essential in developmental and pathological neuronal death (Guo et al., 1998). Levels of Par-4 increase rapidly in response to various apoptotic stimuli through enhanced translation of Par-4 messenger RNA. Par-4 acts at an early stage of the apoptotic cascade prior to caspase activation and mitochondrial dys- function, by a mechanism that may involve inhibition of the antiapoptotic transcription factor NF-k and suppression of Bcl-2 expression and/or function (Mattson et al., 1999). Rai is an Shc-related adaptor protein whose expression is exclusively restricted to the nervous system. It exerts a prosurvival function in neuronal cells by activating the PI3K/Akt signalling pathway (Pelicci et al., 2002). A neuron-specific splice variant of the variable subunit B was characterized, which is induced 89

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upon neuronal differentiation, targets protein phosphatase 2A to mitochondria and accelerates neuronal cell apoptosis after survival factor deprivation (Dagda et al., 2003). Lately, a brain-specific isoform of mitochondrial apoptosis-inducing factor, AIF2, has been isolated (Hangen et al., 2010). AIF2 dimerizes with AIF1 and has a stronger membrane anchorage. Therefore it is conceivable the neuron-specific AIF2 may have been designed to be retained in mitochondria and to minimize its potential neurotoxic activity. In conclusion, although mature neurons are among the most long-lived cell types in mammals, immature neurons die in large numbers and the regulation of apoptosis has a major role in sculpting the developing brain. Furthermore, not only is apoptosis important in regulating neuronal development, but it might also be a cardinal feature in the adult brain in several pathological conditions that will be detailed in the section on neurodegenerative diseases. Role of Magnesium in Neuronal Apoptosis Deciphering the role of magnesium in the apoptotic process is further complicated in the nervous system, as the blood brain barrier may restrain serum magnesium availability within the CNS. Indeed, this has been advocated as the cause for the failure of clinical trials assessing magnesium therapy in acute and chronic brain injury (Vink et al., 2009). A significant part of the literature on the effects of magnesium on apoptosis in the brain is related to perinatal brain, as magnesium is often used as a treatment for pre-eclampsia/eclampsia and preterm labour. The basis for its use as a tocolytic is due to its observed effects in reducing myometrial contractility through extra- and intracellular mechanisms of action (Fomin et al., 2006). However, the use of magnesium sulphate as a drug in obstetrical medicine for both mother and fetus is highly controversial. A recent meta- analysis of all trials has recommended the use of magnesium for neuroprotection in the preterm fetus, as it was both safe and effective (Doyle et al., 2009). Nevertheless, some caution is mandatory, as several reports point to 90

detrimental effects of magnesium for the fetal brain, depending on the dose (Mittendorf et al., 2006; Dribben et al., 2009), on the period of neurodevelopment when the exposure occurred (Dribben et al., 2009) or on the level of stress experienced (Krueger et al., 2001). In addition, some papers report a pro-apoptotic action of magnesium in placental tissues (Black et al., 2001; Gude et al., 2000). The major hurdle to establishing protective or deleterious effects of tocolytic magnesium to the developing brain is that magnesiums effects on preterm infants brains, and the mechanisms of those effects, are not understood. As discussed earlier, neuronal apoptosis can be triggered by three main mechanisms: 1) lack of growth factors; 2) overstimulation of glutamate receptors; and 3) oxidative stress. Magnesium could play a (different) role in each of these signalling pathways. First, most growth factors work through receptor tyrosine kinases (RTKs) which require two magnesium ions for maximal activity. Kinases downstream of these RTKs, such as PI -kinase and the anti-apoptotic kinase Akt require magnesium both for their activation and activity. Therefore, extracellular magnesium could modulate the effect of growth factors on CNS cells, or activate their second messenger systems directly, ultimately affecting viability, proliferation, and apoptosis in these cells. Secondly, under conditions of normal membrane polarization magnesium is known to block the NMDA glutamate receptor and prevent ion flow through the channel. Therefore, in principle, magnesium should protect against excitotoxicity- induced apoptosis. Nevertheless, as already discussed, normal physiological patterns of NMDA receptor activity promote neuroprotection against both apoptotic and excitotoxic insults; vice versa, NMDA receptor blockade can promote neuronal death outright or render neurons vulnerable to secondary trauma (Hardingham, 2009). Indeed, most NMDA antagonists have failed miserably as neuroprotective agents in clinical trials, in large part because of intolerable side effects (Lipton, 2006). Moreover, it has been shown that various drugs that inhibit neuronal activity, including NMDA antagonists, trigger widespread apoptotic neurodegeneration in the

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developing brain and cause long-term neurobehavioral deficits; in the infant rodent brain, peak sensitivity to this effect is in the P3 to P7 period (Ikonomidou et al., 1999). Thus, although other mechanisms may play a role, it seems likely that the neuroapoptogenic action demonstrated for magnesium in some studies could be mediated by its action at NMDA receptors. Third, as mentioned earlier, magnesium deprivation may induce or exacerbate oxidative stress, another trigger of neuronal death, especially in pathological conditions. In this regard, magnesium supplementation might well have a neuroprotective action. Apoptosis in neurodegenerative diseases As already mentioned, neuronal death by apoptosis is a distinctive physiological feature of the developing brain; once synaptogenesis is over, the remaining neurons will usually survive throughout life to preserve brain function. Unfortunately, however, many people experience excessive death of specific neuronal sub- populations as a result of chronic diseases including Alzheimers disease, Parkinsons disease, Huntingtons disease and amyotrophic lateral sclerosis. Neuronal death may also occur as a consequence of acute conditions, such as stroke or traumatic brain and spinal cord injury. For each of these disorders, apoptosis has been implicated as the main form of cell death, though it is very difficult to demonstrate it for both technical and ethical reasons. Much of the evidence supporting an apoptotic mode of neuronal death comes from studies of animal and cell-culture models of neurodegenerative disorders (Mattson, 2000). Physiological apoptosis in the developing brain and pathological apoptosis in the adult brain share similar molecular mechanisms in the effector phase, but may differ in the initiation. Whereas trophic factor withdrawal has a prominent role in apoptosis during development, toxic insults resulting from biochemical or genetic accidents seem to be the triggering event in neurodegenerative disorders. An emerging theme is the toxicity of abnormal protein structures or aggregates (Yuan and Yankner, 2000). Indeed, a defining feature of Alzheimers disease is accumulation of amyloid plaques

formed by aggregates of amyloid- peptide (A). Parkinsons disease is characterized by the appearance of -synuclein oligomers. Huntingtons disease is caused by expansions of a trinucleotide (CAG) sequence in the huntingtin gene producing a protein containing increased polyglutamine repeats. The neuropathological signature of amyotrophic lateral sclerosis (ALS) is the presence of ubiquitinated inclusions immuno- reactive for the proteins TDP-43 and/or FUS/TLS in the cytoplasm of motor neurons. Moreover, mutations in specific genes may predispose to neuronal degeneration, e.g. presenilin in Alzheimer's disease, parkin in Parkinson's disease, huntingtin in Huntington's disease, and Cu/Zn- superoxide dismutase (SOD) in ALS (please refer to the dedicated chapters in this book for further details). The identification of specific genetic and environmental factors responsible for chronic neurodegenerative disorders has bolstered evidence for a shared pathway of neuronal apoptosis involving oxidative stress, perturbed calcium homeostasis and mitochondrial dysfunction ultimately converging on caspase activation (Zndorf and Reiser, 2010; Gibson et al., 2010). In Alzheimer's disease, toxic forms of A may induce Ca2+ influx into neurons by formation of an oligomeric pore in the plasma membrane, thereby rendering neurons vulnerable to excitotoxicity and apoptosis (Supnet and Bezprozvanny, 2010). A also generates hydrogen peroxide and hydroxyl radicals, producing membrane lipid peroxidation and consequently membrane depolarization and excitotoxicity through NMDA receptor channels and voltage- gated Ca2+ channels (Sultana and Butterfield, 2010; Butterfield et al., 2010). In addition, A accumulates in mitochondria, impairs the activity of complexes III and IV of the respiratory chain, causes elevated cytoplasmic Ca2+ levels and oxidative stress, and reduces ATP synthesis, further increasing Ca2+ overload and oxidative stress (Moreira et al., 2010). Mitochondrial dysfunction is a defect occurring early in the pathogenesis of both sporadic and familial Parkinson's disease. Mitochondrial association of -synuclein in cells was linked to impairment of respiratory complex I activity, 91

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oxidative modification of mitochondrial proteins, and increased levels of Ca2+ and nitric oxide (Navarro and Boveris, 2009). Mitochondrial Ca2+ overload is also a decisive commitment step for Huntington's disease. Mutant huntingtin facilitates opening of the PTP and directly impairs the mitochondrial function. Furthermore, huntingtin forms a ternary complex with other proteins, which causes Ca2+ release from the ER and renders neurons more sensitive to Ca2+-mediated cellular dysfunction (Damiano et al., 2010). The mutations in SOD responsible for ALS do not decrease antioxidant activity of the enzyme, but result in the gain of an adverse pro-apoptotic activity that may involve increased peroxidase activity. Through interactions with hydrogen peroxide or superoxide anion, the mutant enzyme may induce oxidative damage to membranes and disturbances in mitochondrial function that make neurons vulnerable to excitotoxic apoptosis (Vucic and Kiernan, 2009). Also, stroke and trauma, and the consequent brain ischemia, initiate biochemical and molecular events involving many of the same neurodegenerative cascades that occur in the chronic neurodegenerative diseases described above, namely excitotoxicity, calcium overload and oxidative stress (Arundine and Tymianski, 2004). In conclusion, despite the concurrence of diverse genetic and environmental factors for acute and chronic neurodegenerative disorders, such conditions possess a common denominator as to the ultimate mechanisms executing neuronal death, which are identical to those involved in normal brain development and all converge to mitochondrial dysfunction. On one hand, these findings testify the evolutionary importance of conserving fundamental processes. On the other hand, they call for further research to understand the basic mechanisms and the key regulators of neuronal fate, as novel opportunities for targeted therapeutic intervention may arise. Role of magnesium in neurodegenerative diseases As detailed above, there is increasing evidence that neuronal death by apoptosis is associated 92

with both acute and chronic neurodegenerative disorders. Interestingly, brain free magnesium levels have been shown to decline in a number of such pathologies (Vink et al., 2009). As a consequence, a considerable research effort has been directed toward establishing the mechanisms of such decline and the potential for magnesium administration as a neuroprotective agent. Brain magnesium decline is a ubiquitous feature of traumatic brain injury and is associated with the development of motor and cognitive deficits. Experimentally, parenteral administration of magnesium up to 12 h post-trauma restores brain magnesium homeostasis and profoundly improves both motor and cognitive outcome. Although the mechanism of action is unclear, magnesium has been shown to attenuate a variety of secondary injury factors, including brain edema, cerebral vasospasms, glutamate excitotoxicity, calcium-mediated events, lipid per- oxidation, mitochondrial permeability transition, and apoptosis. Disappointingly, magnesium therapy has failed in clinical trials. Increase in brain free magnesium concentration seems to be essential to confer neuroprotection, and intravenous magnesium administration only marginally increases CSF magnesium concentration, which suggests that the integrity of the blood- brain barrier and the regulation of magnesium in the cerebrospinal fluid are largely maintained following acute brain injury and limit magnesium bioavailability in the brain (Vink and Nimmo, 2009). Magnesium therapy has also been described in the clinical stroke literature, with similar negative results, though magnesium was shown to be beneficial in a subgroup of patients with noncortical, or lacunar, strokes (Ginsberg, 2008). Magnesium may protect via multiple mechanisms, including NMDA receptor blockade, inhibition of excitatory neurotransmitter release, blockade of calcium channels, as well as vascular smooth muscle relaxation, and may be more bioavailable, as it is well known that the blood brain barrier around infarcted tissue is highly permeable, thus potentially facilitating local magnesium entry to the injured tissues. Intriguingly, the cation channel TRPM7, which is crucial for Mg2+ homeostasis and cell survival (Schmitz et al., 2003), seems to be a critical

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mediator of anoxic cell death. It appears that TRPM7 gating lies downstream of NMDA receptor-mediated NO free-radical production. In this paradigm, excitotoxicity is likely the initiating signal in a cell death cascade that involves nNOS activation with subsequent peroxynitrite production, which in turn activates TRPM7 channels. Calcium influx through TRPM7 then creates a positive feedback loop of ROS production, which eventually kills the cell (Aarts and Tymianski, 2005). TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function; also, it prevented ischemia-induced deficits in long-term potentiation and preserved performance in memory tasks (Sun et al., 2009). The role of the magnesium-specific channel TRPM6 in these processes is currently unclear, but, given its localization in the brain, its permeability to Mg2+ and its functional interaction with TRPM7, it may be involved in neuronal death as well (Cook et al., 2009). At present, the role of TRPM6/7 channels in magnesium transport seems less important than their facilitation of other cation fluxes, but it cannot be ruled out, especially considering the known involvement of magnesium in excitotoxicity and oxidative stress, as discussed earlier. With respect to chronic brain degeneration, the first connection between magnesium and three apparently dissimilar neurodegenerative dis- orders (ALS, Parkinson's and Alzheimer's diseases) came from the observation that those conditions occurred at extraordinarily high rates in geographically separate foci among three genetically different, homogenous western Pacific populations (Durlach et al., 1997). Intensive research conducted over the years led to the identification of two candidate environmental factors: 1) severely low levels of Ca2+ and Mg2+ in the soil and drinking water coupled with abnormal mineral metabolism; and 2) the putative neurotoxin -methylamino-L- alanine (L-BMAA), derived from the cycad plant, a traditional food source in Guam. Experimental findings supported this hypothesis: animals fed diets that mimic the mineral composition in the disease foci environment showed signs of neuronal damage; of the various combinations of Ca2+ and Mg2+ contents tested, exposure to low

Mg2+ (one-fifth of the normal level) was more deleterious, causing significant loss of dopaminergic neurons in the substantia nigra (Oyanagi et al., 2006). Furthermore, motor neurons are selectively more vulnerable to L- BMAA toxicity, and these toxic effects are mediated through [Ca2+]i rises via Ca-A/K channels and other pathways, and subsequent reactive oxygen species generation (Rao et al., 2006). In the hyperendemic foci in the Western Pacific, the unique mineral composition of the environment creates a condition of increased oxidative stress because low Mg2+ levels cause lipid peroxidation while transition metals act as redox catalysts causing increased ROS production. Therefore, the genes whose function is most likely to be affected are ion channels expressed in the CNS and modulated by oxidative stress. TRPM7 and TRPM2 fulfil these conditions; their functional properties are interconnected with calcium and magnesium homeostasis, oxidative stress, mitochondrial dysfunction, and immune mechanisms, all principal suspects in neurodegeneration (Hermosura and Garruto, 2007). Indeed, variants of both genes have been found in Guamanian neurodegenerative disorders and may contribute to their pathogenesis (Hermosura et al., 2008; Hermosura et al., 2005). Despite the evidence implicating magnesium in the aetiology of neurodegenerative diseases, the therapeutic effects of magnesium supplementation are not well documented. Magnesium administration proved beneficial in an in vitro model of Parkinson's disease, where in particular magnesium concentration seems critical in the onset of the disease (Hashimoto et al., 2008), but there is still much to be done to fully appreciate the therapeutic potential of magnesium. Conclusion An extensive and up-to-date review of the literature shows that drawing a clear-cut conclusion on the role of magnesium in neuroapoptosis is not an easy task. Magnesium may play pleiotropic roles in different stages of

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Figure 1. Potential points of interaction between magnesium and apoptotic factors. chronic injuries, blood brain barrier integrity, and the apoptotic process, that range from so on, may concur in determining a pro or anti- modulating growth factor- and Ca2+-dependent apoptotic environment. signal transduction to affecting oxidative stress- related reactions. From this point of view, We believe that further investigation into the role increased extracellular magnesium availability of magnesium in the molecular mechanisms of should in principle be considered anti- apoptosis is warranted for a deeper apoptogenic. On the other hand, an increase in understanding of the basic processes that cytosolic free magnesium, which seems to be regulate cell fate, and ultimately for a successful released from mitochondrial stores, appears to therapeutic intervention when these processes accompany the intrinsic apoptotic process. The go awry. pathophysiological meaning of this occurrence in the context of apoptosis induction/execution awaits to be defined (see Figure 1 for a summary). In a more physiological basis, magnesium Acknowledgments availability in the brain should be considered Supported by the Italian Ministry of Education, within a more complex and interconnected University and Research PRIN 2007ZT39FN and picture, where other conditions, such as Linea D1 2004-2009. developmental s tage, i nflammation, a cute o r

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Brain free magnesium homeostasis as a target for reducing cognitive aging


Jean-Marie Billard *
* jean-marie.billard@inserm.fr

Universit Paris Descartes, Facult de Mdecine Ren Descartes, Centre de Psychiatrie et Neurosciences, UMR 894, Paris, France.


Abstract In the general deterioration of physiological functions that takes place in aging, the prevalence of cognitive impairments, and particularly of those related to learning and memory, makes these deficits a major concern of public health. Although the exact nature of cellular and molecular substrates underlying learning and memory still remains an open issue for the neurobiologist, the current hypothesis assumes that it is determined by the capacity of brain neuronal networks to express short- and long-term changes in synaptic strength. Accordingly, the capacity of functional plasticity is impaired in the brain of aged memory-deficient animals. Short-term changes in synaptic transmission closely depend on transmitter release and neuronal excitability while long-term modifications are mainly related to the activation of the N-methyl-D-aspartate receptor (NMDA-R), a subtype of glutamate receptors. Because transmitter release, neuronal excitability and NMDA-R activation are modulated by magnesium (Mg2+), a change in brain Mg2+ homeostasis could affect synaptic strength and plasticity in neuronal networks and consequently could alter memory capacities. In addition, alteration of brain Mg2+ levels could be regarded as a possible mechanism contributing to cognitive aging. According to these postulates, long-term increase in Mg2+ levels facilitates the conversion of synapses to a plastic state while learning and memory capacities are enhanced in adult animals fed with a diet enriched in Mg2+-L-threonate, a treatment that significantly elevates brain Mg2+ levels. Because Mg2+-L-threonate also improves learning and memory in aged animals, the regulation of brain Mg2+ homeostasis may therefore be regarded as a relevant target for the development of new pharmacological strategies aimed at minimizing cognitive aging. Introduction Alterations in brain anatomy and physiology are frequent features that gradually take place in the course of aging, to finally impair cognitive functions, such as learning and memory. In the last decades, extensive behavioural experiments performed in animal models of aging confirm that the efficient learning and memory in young individuals is slowed down with age, while forgetfulness is accelerated (Barnes and McNaughton, 1985; Gallagher and Rapp, 1997; Lanahan et al., 1997; Norris and Foster, 1999; Ward et al., 1999), and, like in humans, deficits concern several forms of memory, including spatial, associative and long-term memory (Clayton et al., 2002; Gruart et al., 2008; Houston et al., 1999; Rosenzweig and Barnes, 2003; Sykova et al., 2002; Winocur and Moscovitch, 1990; Zornetzer et al., 1982). Neuronal or synaptic loss is unlikely to significantly account for the senescent-associated cognitive deficits (Eriksen et al., 2009; Geinisman et al., 2004; Luebke et al., 2010; Morrison and Hof, 2007; Rapp and Gallagher, 1996) and a wealth of data now rather indicate that changes in functional properties within neuronal networks are mainly concerned (Billard 2006; Burke and Barnes, 2006; Craik and Bialystok, 2006; Disterhoft et al., 1994; Driscoll et al., 2003; Erickson and Barnes, 2003; Foster, 2007; Grady and Craik, 2000; Hsu et al., 2002; Kelly et al., 2006; Lister and Barnes, 2009; Magnusson, 1998; Mora et al., 2007; Sykov et al., 1998; Toescu and Verkhratsky, 2004). In particular, studies of long-lasting modifications of glutamatergic neurotransmission, such as long- term potentiation (LTP) and long-term depression (LTD) of synaptic strength, now considered as functional substrates of memory encoding (Bear and Malenka, 1994; Bliss, 1990; Collingridge and Bliss, 1995; Eichenbaum, 1996; Izquierdo, 1991; Kim and Linden, 2007; Lisman and McIntyre, 99

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2001; Martin et al., 2000; Teyler and DiScenna, 1987), show substantial changes with age (Barnes, 2003; Billard, 2006; Burke and Barnes, 2006; Foster, 2006; Norris et al., 1996). Among the different mechanisms that may account for these age-associated impairments of synaptic plasticity (Foster, 2007; Rosenzweig and Barnes 2003), the activation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDA-R) has received particular attention. Indeed, these receptors are pivotal for the regulation of synaptic strength, by means of their high permeability to calcium (Ca2+), which triggers the activation of specific intracellular protein kinases and phosphatases (Wang et al., 1997). Although it is obvious that NMDA-R activation is impaired in aging (Barnes et al., 1997; Junjaud et al., 2006; Potier et al., 2000), all of the underlying mech- anisms have yet to be definitely characterized and it remains to be determined to what extent they are involved in age-related deficits in synaptic plasticity and of memory capacities. Among the different mechanisms possibly involved in cognitive aging is a change in ion homeostasis (Roberts, 1999) since transmitter release, cellular excitability and expression of synaptic plasticity closely depend on ion flux across neuronal membranes. Although Ca2+ regulation has initially gathered the largest interest in aging studies (Thibault et al., 2007; Toescu et al., 2004; Verkhratsky and Toescu, 1998), the role of magnesium (Mg2+), which is found in a relative large concentration in the central nervous system (CNS) (Chutkow, 1974; Poenaru et al., 1997), is now much more considered. Indeed, aging is a risk factor for Mg2+ deficit (Wakimoto and Block, 2001) [for a review see (Durlach et al., 1998)] and brain Mg2+ levels are significantly reduced in age- associated neurodegenerative diseases (Andrasi et al., 2000; Andrasi et al., 2005; Basun et al., 1991). On the other hand, the well-known regulation of NMDA-R activation by Mg2+ (Mayer and Westbrook, 1987; Nowak et al., 1984) and the fact that altered Mg2+ levels impair memory functions (Bardgett et al., 2005; Bardgett et al., 2007; Landfield and Morgan, 1984), strongly suggest that a change in Mg2+ homeostasis could contribute to the physiopathology of cognitive aging. After reviewing the different roles of Mg2+ in the regulation of synaptic mechanisms at glutamat- 100

ergic synapses, the present report will consider whether Mg2+ could be involved in deficits of these mechanisms that occur in the aging brain, and, finally, recent data will be presented suggesting Mg2+ as a relevant dietary component that could help in reducing age-associated memory impairments. The impact of brain Mg2+ levels on synaptic mechanisms contributing to cognitive functions Although Mg2+ is the second most abundant intracellular mineral after potassium and is present at large amount in the cerebrospinal fluid of both rodents (0.8 mM) (Chutkow, 1974) and humans (1.0 to 1.2 mM) (Basun et al., 1991; Kapaki et al., 1989), its role on neural activity and synaptic plasticity has been much less considered compared to other divalent cations such as calcium (Ca2+). This is rather surprising considering that Mg2+ is a cofactor for more than 300 enzymes and also tightly interacts with phospholipids and nucleic acids (Hofmann et al., 2000; Wolf and Cittadini, 2003), suggesting that the mineral should be able to modulate brain activity on a broad scale. Initially, the role of Mg2+ has mainly been evaluated in vitro by lowering extracellular levels ([Mg2+]e), a procedure that increases spontaneous firing rate of neurons through membrane depolarization (Furukawa et al., 2009; Stone et al., 1992). This decrease in [Mg2+]e can lead to paroxysmal events in slice preparations from both animals and humans, which resemble abnormal activities occurring during sustained seizures in vivo (Armand et al., 1998; Jones and Heinemann, 1988; Stanton et al., 1987). Using high performance liquid chroma- tography, quantification of basal efflux of amino acids indicates that only levels of glutamate, the neurotransmitter involved in most of excitatory synapses in the CNS, are significantly enhanced in low [Mg2+]e medium (Furukawa et al., 2009; Smith et al., 1989). These data point out a first contribution of Mg2+ regarding the activity of excitatory synapses in the brain, that is, the regulation of the probability of transmitter release (Figure 1, left), as initially characterized at neuromuscular junctions (Kelly and Robbins, 1983; Kuno and Takahashi, 1986). But several lines of evidence show that beside this ubiquitous control on the probability of glutamate release at presynaptic terminals, Mg2+is also able to influence

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Figure 1. Schematic representation of the positive and negative effects of short-term (left) and long-term (right) elevation of brain Mg2 levels on neurotransmission and synaptic plasticity at an excitatory glutamat- ergic synapse. AMPAr: -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; NMDAr: N-methyl- D-aspartate receptor, VGCC: voltage-gated calcium channels. through the regulation of AHP amplitude and synaptic activity by acting at postsynaptic level duration, Mg2+ is able to modulate synaptic (Figure 1, left). For instance, frequency potentiation (FP), which represents an increase in strength and to alter cognitive abilities synaptic strength rapidly developing during (Disterhoft and Oh, 2006). repetitive activation of glutamatergic afferent fibres (Anderson and Lomo, 1966; Andreasen and Although there is no doubt that Mg2+ may be considered as firmly involved in the regulation of Lambert, 1998), is greater after elevating [Mg2+]e synaptic activity through the pre- and post- (Landfield and Morgan, 1984). This is mainly due 2+ synaptic mechanisms described above, an to the ability of Mg to reduce the calcium- additional and essential role for the mineral has dependent post-burst after hyper-polarization emerged at the end of the 1980s from (AHP) of membrane potential, which is normally electrophysiological studies demonstrating that induced in depolarized neurons to limit excessive Mg2+ inhibits currents through channels associated firing (Hotson and Prince, 1980; Lorenzon and with the N-methyl-D-aspartate subtype of gluta- Foehring, 1992; Madison and Nicoll, 1984). AHPs mate receptors (NMDA-R) by directly blocking the are reversibly reduced by the acquisition of ion pore (Bekkers and Stevens, 1993; Jahr and learning-dependent behavioural tasks such as Stevens, 1990; Mayer and Westbrook, 1987; trace eye blink conditioning or spatial water maze (Moyer et al., 2000; Thompson et al., 1996). Thus, Nowak et al., 1984). A transient rise in [Mg2+]e by controlling the initial postsynaptic depolarization within physiological range, i.e. from 0.8 to 1.2 101

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mM, rapidly reduces the current amplitude mediated by NMDA-R by more than 60% in cultured neurons clamped at potentials below -50 mV while further increase in [Mg2+]e is significantly less effective (Slutsky et al., 2004). Importantly, this depression effect does not occur at depolarized potentials, revealing a voltage dependency for the block of NMDA-R by Mg2+, which underlies the pivotal role of NMDA-R in the induction of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Indeed, following high-frequency activation of presynaptic terminals, the glutamate-induced depolarization of membrane potential releases NMDA-R from Mg2+ block allowing Ca2+ to enter the cells (Figure 1, left). As a consequence, activation of specific kinases or phosphatases is triggered, thus modifying synaptic strength (Wang et al., 1997). Several lines of evidence show effects of transient changes in Mg2+ levels on the induction of synaptic plasticity. For instance, a rapid rise in [Mg2+]e selectively antagonizes LTP by reducing the depolarization- induced Ca2+ influx (Dunwiddie and Lynch, 1979; Malenka et al., 1992; Malenka and Nicoll, 1993). Interestedly, LTP is also suppressed in slices bathed with a Mg2+-free medium (Frankiewicz and Parsons, 1999; Jouvenceau et al., 2002), a loss that is independent of NMDA-R activation but rather due to changes in signalling cascades in post-synaptic neurons that remain to be characterized (Hsu et al., 2000). At intracellular level, Mg2+ also regulates the activity of Ca2+- dependent protein kinases governing NMDA- dependent LTP. For instance, it controls the subcellular localization of protein kinase C (PKC), which closely determines the function of the enzyme (Tanimura et al., 2002) and stimulates the dephosphorylation and deactivation of Calmodulin Kinase II (CaMKII) (Easom et al., 1998). Despite much of data underlining that a transient increase in Mg2+ levels unables synapses to remain highly plastic, an unexpected opposite result has been reported in vitro after studying the effects of long-term elevation of the mineral (Figure 1, right). Indeed, when [Mg2+]e is increased within physiological range in neuronal cultures for more than several hours, NMDA-R-mediated currents are enhanced and the expression of LTP significantly facilitated (Slutsky et al., 2004). In the same conditions, synaptic strength is not 102

modified following a single action potential but increased after a burst of inputs. These long-term facilitation effects of Mg2+ on neurotransmission and synaptic plasticity also occur in vivo since they are found in young rats fed with a diet enriched in Mg2+-L-threonate (Mg2+-T), a new highly bioavailable compound that enhances loading of Mg2+ into the brain (Slutsky et al., 2010). The increase in the density of functional presynaptic boutons (Slutsky et al., 2010) coupled with the weaker glutamate release at synapses found after chronic Mg2+ elevation (Slutsky et al., 2004) indicate that long-lasting changes in Mg2+ homeostasis are able to modify the pattern of synaptic assemblies within neuronal networks, from a limited number of synapses with high probability of release to a larger density with low probability of release (Figure 1, right). In addition, both the increase in [Mg2+]e in vitro and the elevation of brain Mg2+ in vivo up-regulate the expression of NR2B-containing NMDA-R (Slutsky et al., 2004; Slutsky et al., 2010). This increase, proposed to counterbalance the higher blockade of NMDA-R opening associated with chronic elevation of [Mg2+]e, contributes to the greater capacity of synapses to be highly plastic. Because the pattern and strength of synaptic transmission are widely believed to code memory traces (Bear and Malenka, 1994; Bliss, 1990; Collingridge and Bliss, 1995; Eichenbaum, 1996; Izquierdo, 1991; Kim and Linden, 2007; Lisman and McIntyre, 2001; Martin et al., 2000; Neves et al., 2008; Teyler and DiScenna, 1987), their susceptibility to short- and long-term changes in Mg2+ homeostasis described above predict that cognitive abilities would also be modulated by altering Mg2+ levels. Accordingly, Mg2+ deficiency impairs fear-conditioning (Bardgett et al., 2005; Bardgett et al., 2007) while chronically elevating plasma Mg2+ over several days improves reversal learning in the hippocampus-dependent T-maze task (Landfield and Morgan, 1984). However, whether brain Mg2+ levels are really altered in these studies has been questioned considering that Mg2+ loading into the brain is tightly regulated by active transport processes that maintain a concentration gradient between the cerebrospinal fluid (CSF) and the plasma. In fact, Mg2+ levels are poorly affected in the brain after long-lasting increase in plasma Mg2+ induced by intravenous injection of MgSO4 both in animals and humans (Kim et al., 1996; McKee et al., 2005).

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Nevertheless, this rigorous control of brain Mg2+ levels has recently been overcome using the highly bioavailable compound Mg2+-T, which increases Mg2+ concentrations in CSF by at least 15% (Slutsky et al., 2010). Behavioural characterization of the Mg2+-T-treated rats indicates significant improvements of learning abilities, working memory as well as short- and long-term memory compared to control animals (Slutsky et al., 2010), confirming that even moderate, changes in brain Mg2+ homeostasis are capable of altering cognitive performances. The impact of brain Mg2+ levels on impaired synaptic mechanisms underlying cognitive aging Although a link between Mg2+deficiency and cellular senescence has been proposed for the age- related deterioration of a large range of physiological functions (Killilea and Maier, 2008), a causal effect on deficits of cognitive functions is still questioned. Even aging is thought of as a general risk factor for Mg2+ deficit (Durlach et al., 1993; Wakimoto and Block, 2001). In fact, Mg2+concentrations determined in various brain structures only slightly decrease in healthy aging (Morita et al., 2001; Takahashi et al., 2001). However, since even a very small disturbance of [Mg2+]e is able to substantially modify synaptic assemblies supporting cognitive performances as reported above, it may be postulated that age- related changes in Mg2+ levels, even of very weak amplitude, could contribute to the physio- pathology of cognitive aging. Regarding the regulation of transmitter release at presynaptic terminals, the increase in basal release of glutamate determined in the brain of aged rats and monkeys in vivo (Massieu and Tapia, 1997; Quintero et al., 2007) suggests a possible role for Mg2+. According to this postulate, spontaneous miniature end-plate potentials increase in amplitude at neuro- muscular junctions in aging, that has been shown to reflect an impaired regulation of transmission release by Mg2+ (Kelly and Robbins, 1983). Nevertheless, whether a similar effect of Mg2+ also occurs at central synapses still remains to be demonstrated. Age-related decrease in neuronal excitability is well documented, indicating that the amplitude and duration of AHPs are enhanced with age

(Disterhoft and Oh, 2006; Disterhoft and Oh, 2007; Power et al., 2002; Thibault et al., 2007). AHP amplitude inversely correlates with both acquisition and probe performance in learning behaviours among aged animals (Tombaugh et al., 2005) and pharmacological treatments that rescue the age-related alteration of AHPs, also minimize memory deficits (Oh et al., 1999; Weiss et al., 2000). These data strongly suggest that a decrease in neuronal excitability is a potent mechanism contributing to the physiopathology of cognitive aging (Disterhoft and Oh, 2006). Although the involvement of Mg2+ in this functional deficit has not yet been formally demonstrated, some indirect experimental data suggest that this is probably the case. Frequency potentiation, which is negatively correlated to AHP magnitude (Thibault et al., 2001), is reduced by age (Diana et al., 1994; Landfield and Lynch, 1977; Landfield et al., 1986; Thibault et al., 2001) and this alteration is prevented by elevating [Mg2+]e (Landfield and Morgan, 1984; Landfield et al., 1986). From these results, it may be hypothesized that the age-related facilitation of Ca2+ conductances supporting the increase in AHPs is not only due to a greater density of Ca2+ channels on neuronal membranes (Campbell et al., 1996; Thibault and Landfield, 1996; Thibault et al., 2001; Veng et al., 2003) but also to some extent, to a weaker competition between cations following Mg2+ depletion. Regarding the expression of long-lasting synaptic plasticity, extensive electrophysiological studies report age-related deficits of both LTP and LTD in aged memory-impaired animals that reflects a shift in Ca2+ sources, with a weaker role for NMDA-R, and an increased contribution of voltage- gated Ca2+ channels and intracellular stores with different kinetic properties (Foster and Kumar, 2002; Gant et al., 2006; Junjaud et al., 2006; Kumar and Foster, 2005; Shankar et al., 1998; Thibault et al., 2007). NMDA-R activation is impaired in aged animals (Barnes et al., 1997; Burke and Barnes, 2010; Clayton et al., 2002; Eckles-Smith et al., 2000; Fontan-Lozano et al., 2007; Kollen et al., 2010; Magnusson, 1998; Ontl et al., 2004; Potier et al., 2000), not because of a reduced receptor density but rather to changes in pharmacological properties of the glutamatergic receptor (Billard and Rouaud, 2007; Junjaud et al., 2006; Kollen et al., 2010; Kuehl-Kovarik et al., 2003; Mothet et al., 2006; Turpin et al., 2009). 103

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Among the possible mechanisms affecting NMDA-R activation in aging, a role for Mg2+ has been evaluated. No significant alteration of NMDA-R susceptibility to Mg2+ block occurs during aging since the percent decrease in NMDA-R- mediated synaptic potentials is comparable in young and aged animals after transient [Mg2+]e elevation (Barnes et al., 1997; Potier et al., 2000). Also altering [Mg2+]e affects NMDA-R-dependent LTP as well as short-term potentiation (STP) in a similar way in the two groups of animals indicating that a change in Mg2+ block of NMDA-R is unlikely to contribute to the age-related impairment of synaptic plasticity (Potier et al., 2000). However, a role for Mg2+ in LTP deficits occurring in aged animals does not definitively be discarded. For instance, a weaker activation of NMDA-R by its co-agonist D-serine has been shown to underlie LTP impairment in aged rodents, that is due to a weaker production of the amino acid by its synthesizing enzyme serine racemase (SR) (Junjaud et al., 2006; Mothet et al., 2006; Potier et al., 2010; Turpin et al., 2009). Because SR activity is potently stimulated by Mg2+, which increases 5- to 10-fold the rate of racemization of L- to D-serine, an impaired activation of SR by Mg2+ may therefore be possibly regarded as a potent mechanism linking lower brain Mg2+ levels to deficits of NMDA-R activation and related synaptic plasticity in aging. Regarding the effects of long-lasting elevation of [Mg2+]e, functional improvements also occur at synapses of the aging brain (Slutsky et al., 2010). The density of synaptophysin- and synaptobrevin- immunostained puncta is decreased in the hippocampal dentate gyrus of aged animals whereas synaptic loss in the stratum radiatum of CA1 subfield is less pronounced and even remains controversial (Burke and Barnes, 2006; Geinisman et al., 2004; Smith et al., 2000). However, in aged Mg2+-T treated rats, the number of functional synaptic connections is significantly increased in both hippocampal areas compared to aged controls (Slutsky et al., 2010). As in young animals, a treatment with Mg2+-T also up-regulates NR2B- containing NMDA-R, thus improving LTP expression in slices from aged animals (Slutsky et al., 2010). Interestingly, both the increase in functional synaptic connections and the facilitated induction of synaptic plasticity (Figure 2A) (see (Slutsky et al., 2010)). In addition, it is worth noting in these aged treated animals that 104

Figure 2. Elevation of brain Mg2 levels by Mg2+-T improves memory in aged rats. (A). Mg2+-T aged treated rats show shorter escape time in the water maze task indicating faster learning. (B). Time course of the reversible benefit effect of Mg2+-T treatment on spatial working memory in aged rats. Modified from (Slutsky et al., 2010). the control level after the end of Mg2+-T supplementation and the time course of this decrease matches that of the reinstallation of impairment in memory scores (Figure 2B). Other possible routes involving brain Mg2+ in cognitive aging The basal forebrain cholinergic complex including the medial septum/diagonal band, the substantia innominata, and the nucleus basalis of Meynert of Broca, represents a second major excitatory pathway of the CNS (Dutar et al., 1995; McKinney et al., 1983; Mesulam et al., 1983; Mesulam et al., 1984; Woolf et al., 1984). During the past fifty years, many experiments were directed at testing

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whether this cholinergic pathway is involved in aspects of cognition and a significant role in learning and memory has been firmly debated (see (McKinney and Jacksonville, 2005) for a review). Electrically-induced release of acetyl- choline generates slow and long-lasting excitatory postsynaptic potentials involving the activation of muscarinic receptors (Dutar and Nicoll, 1988; Misgeld et al., 1989; Muller and Misgeld, 1986). These responses are impaired in aging (Lippa et al., 1985; Potier et al., 1993; Segal, 1982; Shen and Barnes, 1996; Taylor and Griffith, 1993), that reflects a decreased transmitter release from cholinergic terminals (Aubert et al., 1995; Birthelmer et al., 2003; Scali et al., 1994; Takei et al., 1989; Vannucchi et al., 1997) but also a weaker activation of postsynaptic receptors (Calhoun et al., 2004; Lippa et al., 1985; Potier et al., 1992; Potier et al., 1993; Segal, 1982; Vannucchi et al., 1997). However, it is worth noting that the degree of memory deficit does not appear to closely correlate with changes in cholinergic synaptic activity (Calhoun et al., 2004; Potier et al., 1993; Shen and Barnes, 1996), suggesting that acetylcholine should not play a critical role in cognitive aging as initially proposed (Bartus et al., 1982; Bartus et al., 1985; Pepeu, 1988). In addition to reducing acetylcholine release (Jope, 1981; Vickroy and Schneider, 1991), Mg2+ also acts on postsynaptic muscarinic receptors through several distinct mechanisms. First, Mg2+ is able to bind to the allosteric region of the M2 subtype of receptors, decreasing the inhibitory effects of some modulators on the dissociation of orthosteric ligands (Burgmer et al., 1998). Second, Mg2+ reduces a non-selective cationic conductance activated by muscarinic agonists

(Guerineau et al., 1995), and, finally, the cation is necessary for activating G-proteins coupled to muscarinic receptors (Cladman and Chidiac, 2002; Shiozaki and Haga, 1992; Zhang et al., 2004). Regarding these multiple interactions, it is obvious that changes in Mg2+ levels are likely to contribute to the alteration of cholinergic neurotransmission that occurs in aging. Although direct evidence is still lacking, several data of the literature argue for the possibility that changes in brain Mg2+ may contribute to the age-associated impairment of acetylcholine-dependent synaptic activity. For instance, hypomagnesia significantly weakens responses of cortical neurons to ion- tophoretically applied acetylcholine (El-Beheiry and Puil, 1990) while the high-affinity binding at the muscarinic receptor in Alzheimers disease is closely regulated by Mg2+ (Ladner and Lee, 1999). Conclusion Although it is obvious that additional studies are necessary to unravel the mechanisms connecting brain Mg2+ homeostasis and cognitive aging, experimental data has progressively accumulated showing that even moderate changes in the concentration of the mineral are able to significantly affect the assembly and functionality of neuronal networks involved in cognition. Recently, the World Health Organization reached consensus that in a majority of the world's population, the dietary Mg2+ intake is lower than recommended, especially in the aging population (see also (Ford and Mokdad, 2003; Galan, 1997). Based on the results summarized in the present review, there is no doubt that elevating Mg2+ levels in the brain of the elderly could represent a promising strategy to minimize or even prevent cognitive deficits that take place with age.
Andrasi E, Pali N, Molnar Z, Kosel S (2005) Brain aluminum, magnesium and phosphorus contents of control and Alzheimer-diseased patients. J Alzheimers Dis 7:273-84. Andreasen M, Lambert JD (1998) Factors determining the efficacy of distal excitatory synapses in rat hippocampal CA1 pyramidal neurones. J Physiol 507 (Pt 2):441-62.

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Section 2 Magnesium in Neurological Diseases



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The role of magnesium therapy in learning and memory


Michael R. Hoane *
Restorative Neuroscience Laboratory, Brain and Cognitive Science Program, Department of Psychology, Southern Illinois University, Carbondale, IL, USA.
* mhoane@siu.edu

Abstract The old saying you are what you eat is becoming increasingly important in the field of neuroscience these days. There is mounting evidence that nutritional factors are beginning to play a major role in cognitive status, or cognitive wellbeing. One of these emerging factors is magnesium (Mg2+). Although the physiological investigation of Mg2+ has a long history, its role in cognitive function is just starting to emerge. The focus of this chapter is to review the available literature on the effects of Mg2+ on cognitive function in the healthy and diseased/injured brain. In addition, data from our laboratory will be presented that has investigated the effects of Mg2+ manipulation on learning and memory tasks in rodents, as well as the ability of Mg2+ therapy to improve cognitive performance in the damaged brain.

Introduction The purpose of this chapter is to provide a review of the literature on the role of Mg2+ in cognitive function. Although the research on this topic is generally sparse, there is an accumulating body of evidence suggesting that Mg2+ is vitally important. The role that micronutrients play in maintaining and promoting cognitive ability and neural plasticity has started to receive a good deal of attention. A recent paper has provided an excellent review on how various nutrients promote cognitive performance and neural plasticity (Gmez-Pinilla, 2008). Although this review does not specifically discuss the role of Mg2+, it is clear that this is a rapidly evolving area of research. Another review does address the role of Mg2+ and its relationship with other micronutrients on cognitive function and performance. It especially highlights the important role that Mg2+ has in interacting with other micronutrients (Huskisson et al., 2007). Recent evidence also suggests that Mg2+ plays an important role in age-related deficits in neurotransmitter release, neuronal excitability and synaptic plasticity related to cognition (Billard, 2006), and these topics will be reviewed in Chapter 6. Additionally, there is ample evidence that Mg2+ plays an important role in the pathophysiological processes following traumatic brain injury (TBI) and that Mg2+ therapy is effective in promoting

functional recovery in a variety of animal models (Hoane and Barth, 2001; Hoane, 2004; Sen and Gulati, 2010; van den Heuvel and Vink, 2004; Vink et al., 2009). Specifically, this chapter will focus on reviewing the literature on Mg2+ and cognitive function and review a series of studies conducted in our laboratory that have investigated the ability of Mg2+ therapy to alter cognitive function and to improve cognitive recovery following focal and traumatic brain injuries in the rat. Introduction to rodent cognitive assessment The Morris water maze (MWM) is a standard task for measuring cognitive/spatial performance in rodents. This task uses a water-filled tank with a submerged escape platform and many different aspects of memory can be assessed (Hoane et al., 2003; Hoane, 2005; Hoane et al., 2009; Kaufman et al., 2010; Lindner, 1997; Lindner et al., 1998; Quigley et al., 2009). Briefly, a reference memory trial consists of placing the rat into the water at one of 4 randomly chosen start locations. A computer-assisted video tracking system is used to measure the swim latency and distance to the submerged escape platform. On trials designed to measure working memory the escape platform is relocated to a new position in the tank every day. The first trial (in the new location) is considered an information trial and the subsequent 3 trials are test trials and are averaged to form the dependent variable. Additional paradigms can be used that 115

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incorporate various mazes or dividers that are placed into the tank that can test other aspects of learning and memory in the rodent. Additionally, the Barnes spatial maze, a dry version of the MWM, can also be used to assess cognitive/spatial performance (Vink et al., 2003). Rats are placed on an elevated 1.2 m circular tabletop with 19 holes cut around the periphery. The rat learns the location of the hole that leads to an escape tunnel. The latency to find the correct hole is then analysed. Operant chambers (Skinner boxes) can be used to assess appetitive conditioning of learning ratios under various conditions. In general, rats are shaped to bar- press for a reinforcer and then are shifted to more complicated reward paradigms. Thus, there are many ways in which rodent cognitive assessments can be made and many of these have been utilized to examine the role of Mg2+ in cognition. Role of Mg2+ in health and cognition

The importance of Mg2+ in normal cellular functioning has been well documented, as has its importance in the pathophysiology following injury. Previously, several reviews have been written that address these issues (Hoane and Barth, 2001; Hoane, 2004; van den Heuvel and Vink, 2004) so only a brief synopsis will be provided in this paper. Mg2+ is involved in many critical cellular processes including cellular respiration, protein synthesis, membrane stability and regulation of vascular tone. A more detailed discussion on the physiology of Mg2+ is presented in several chapters within Section 1 of this book. Although the main focus of this chapter is on animal models, there are some interesting human studies that have examined the role of Mg2+ in cognitive ability. A recent study examined the correlation between levels of several trace minerals (iron, Mg2+, potassium and zinc) in the hair of adolescent girls and their academic record. Although care must be taken with the interpretation of correlational studies of this nature, there was evidence that some trace minerals correlated more highly with increased academic performance. Specifically, it was found that Mg2+ and zinc demonstrated a strong positive correlation with academic performance (Wang et al., 2008). Furthermore, a recent case 116

report of a patient presenting with anorexia nervosa and Wernicke-Korsakoff syndrome was found to have a low serum Mg2+ level (Saad et al., 2010). Although the main cause of this condition was thiamine deficiency, a major adjunct factor was believed to be Mg2+ deficiency. The results from this case study are supported by a review article on micronutrients and cognitive performance that details the inter- relationships between Mg2+ and other micronutrients such as the B-group vitamins and how deficiencies in these nutrients interact to produce cognitive deficits (Huskisson et al., 2007). Thus, there is an expanding literature that suggests that Mg2+ status plays an important role in cognitive performance. A unique condition in which Mg2+ has been implicated is TBI. McIntosh and colleagues have shown that Mg2+ homeostasis is disrupted following CNS injury (McIntosh et al., 1988; Vink et al., 1988). Fluid percussion injury (FPI) produced a rapid and severe decline in intra- and extracellular Mg2+ levels, which correlated significantly with the severity of the behavioural deficits observed following injury (McIntosh et al., 1988; Vink et al., 1988). Additional research on the role of Mg2+ and neurological diseases will be presented in Section 2 of this book, while Mg2+ in cognitive function following TBI is addressed later in this chapter. These findings suggest that Mg2+ plays an important role in normal physiology and in the pathophysiological events that occur following injury to the nervous system. Recent laboratory data Mg2+ therapy and learning From a purely pharmacological standpoint, administration of Mg2+ immediately prior to the acquisition of a learning task should have a detrimental effect on that task given the non- competitive antagonistic properties of Mg2+ at the NMDA receptor. It has been shown that administration of other NMDA antagonists such as MK-801 and PCP have been shown to disrupt spatial learning in rodents (Kesner et al., 1993; McLamb et al., 1990; Murray and Ridley, 1997); however, in some cases a facilitative effect can be shown (Pussinen and Sirvio, 1999). In order to examine the biological activity of Mg2+ administration on the acquisition of learning, a MgCl2 solution was administered (1 mmol/kg or 2

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mmol/kg, i.p.) prior to the acquisition of learning a reference memory task in the MWM (Hoane, 2007). Intact rats received daily injections of MgCl2, 30 min prior to the start of their MWM session and were tested for 5 consecutive days for 4 trials per day with an intertrial interval (ITI) of 15 min. The reference memory acquisition paradigm was used in the MWM. In this paradigm, the submerged escape platform stayed in the same location for every trial and the animals were released from 1 of 4 different starting points in the maze. As can be seen in Figure 1, the vehicle-treated (0.9% saline, i.p.) group showed steady acquisition of the task. In comparison, the MgCl2-treated animals showed a more varied response. The initial acquisition of the 2 mmol group was slightly lengthened on day 1 compared to the other groups. On day 4, both groups of MgCl2-treated animals started to show a lengthening of their escape latencies compared to vehicle-treated animals. On the last 2 test days the comparison of swim latencies between the 2 mmol and saline group was significantly different (p < 0.01) indicating a possible learning impair- ment on the task. Thus, it appears that daily dosing with the higher dose of MgCl2 produced a significant degree of amnesia after 3 days. Unfortunately, testing was terminated after 5 days so it is unknown if this effect would have persisted or worsened with additional testing. It should be noted that, in general, the NMDA antagonists that work at the PCP site on the NMDA receptor (i.e., MK-801 and PCP) seem to have a greater amnesic effect than MgCl2. This may raise some concerns for continued dosing regimens of Mg2+ therapy lasting more than a couple days. However, a longer window of time between administrations and testing may reveal different findings, because this effect is perceived to be state dependent. Thus, given these behavioural results it is clear that systemic injections of MgCl2 did indeed exert behavioural effects in uninjured animals with an intact blood-brain barrier (BBB), and therefore give support to the ability of MgCl2 to cross the BBB. This finding supports earlier research to this fact (Hallack et al., 1992). We have also recently investigated the effect of dietary Mg2+ deficiency on learning acquisition in the MWM. Rats were placed on either a standard laboratory diet or a commercially available Mg2+-

Figure 1. The effects of repeated pre-testing administration of MgCl2 (1 or 2 mmol/kg) on the acquisition of a reference memory task in the MWM. Plotted are the mean ( SEM) swim latencies to find the submerged escape platform. The 2 mmol dose of MgCl2 significantly impaired swim latencies starting on the 4th day of testing (* = p < 0.05). deficient diet for 14 days. Peripheral blood collect- ions were performed for determination of serum Mg2+ levels and all animals were placed back on to the standard diet. The serum analysis indicated a significant loss in serum Mg2+ in the deficiency group (p < 0.05). One week later, animals were tested for the acquisition of a reference memory task over 4 days (4 trials per day, 15 min ITI). As can be seen in Figure 2A, the 14 days of Mg2+ deficiency significantly impaired the initial acquisition of the task on the first day (p<0.05). The animals were then switched to a more challenging working memory assessment in the MWM. As can be seen in Figure 2B, the animals that experienced Mg2+ deficiency showed impaired learning on the task (p<0.05). Additionally, animals were trained to acquire a fixed ration (FR-10) schedule of reinforcement in an operant chamber. The total number of bar presses is shown in Figure 2C. The higher number of responses results in a greater number of reinforcers being delivered and can be equated to the learning of the reinforcement schedule. Animals on the Mg2+-deficient diet responded at a significantly lower level (p<0.05) than those animals on the Mg2+-normal diet, suggesting a learning impairment. A somewhat similar finding has been shown in mice exposed to a Mg2+-deficient 117

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Figure 2. The effects of dietary restriction of Mg2+ on cognitive performance in the MWM. Plotted are the mean swim latencies ( SEM) on the acquisition of reference memory task (A), working memory (B), and the acquisition of a FR-10 reinforcement schedule in an operant chamber (C). A two-week period of dietary Mg2+ deficiency, followed by reinstatement back to a Mg2+-normal diet resulted in significant impairments in cognitive performance on all measures (* = p < 0.05). diet (Bardgett et al., 2005; Bardgett et al., 2007). In animal models of TBI. It has been shown that 2+ this study, 10 days of Mg deficiency resulted in administration of MgCl2 following FPI improved impaired learning of a conditioned fear task. cognitive outcome by reducing memory loss However, maze performance in the MWM was following injury (Smith et al., 1993). However, not impaired. Thus, these data suggest that even administration of MgCl2 failed to improve the temporary Mg2+ deficiency may interfere with acquisition of a reference memory task in the MWM following injury (Bareyre et al., 1999). It learning. has also been recently shown that administration of MgSO4 (250 mol/kg, i.v.), 30 min following A newly formulated form of Mg2+, magnesium-L- threonate (MgT) has been shown to increase diffuse axonal injury improved recovery of a cerebrospinal fluid levels of Mg2+ to a much spatial memory task on the Barnes maze (Vink et al., 2003). In addition, administration of an greater degree than other Mg2+ preparations (e.g. intravenous solution of MgCl2 (150 mol) prior to MgCl2) (Slutsky et al., 2010). Administration of MgT has also been shown to result in an FPI prevented the occurrence of injury-induced enhancement of various forms of cognitive/ impairments on working and reference memory learning ability in rodents, including working tasks in a radial maze (Enomoto et al., 2005). memory. This effect was also evident in aged rats following which cognitive abilities were generally There have been very few attempts to examine reduced. The beneficial behavioural effects were the ability of Mg2+ therapy to resolve long-term thought to be caused by beneficial changes in cognitive dysfunction. A recent study has shown synaptic plasticity within the hippocampus severe cognitive deficits in the acquisition of a (Slutsky et al., 2010). reference memory task in the MWM when tested 8 months post-FPI (Browne et al., 2004). The 2+ Effects of Mg therapy on cognitive function administration of MgSO4 (125 mol, i.v.) or NPS following brain injury 1506 (an NMDA antagonist) failed to improve the Several studies have investigated the ability of acquisition of the reference memory task 2+ Mg to improve acute cognitive function in compared to vehicle controls. However, it was 118

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found that MgSO4 did reduce the amount of ipsilateral hippocampal cell loss. Thus, preserv- ation of the hippocampus failed to result in significant cognitive improvement (Browne et al., 2004). Given the limited number of studies and their mixed results, it is important to further investigate the effect of Mg2+ therapy on the recovery of cognitive function following injury. Examination of Mg2+ therapy following focal injury In our laboratory we have examined recovery of cognitive function in a bilateral focal cortical ablation model. Rats were given small (4 mm2) electrolytic lesions aimed at the bilateral anterior medial cortex (bAMC) of the frontal lobe (Hoane et al., 2003). Administration of Mg2+ therapy occurred 15 min following injury with rats receiving either injections of MgCl2 (1 or 2 mmol/kg, i.p.) or saline (1 ml/kg). This regimen was repeated again 24 and 72 hrs later, so that each rat received 3 injections within the first 72 hrs following injury. Behavioural testing began 5 days after injury and included the assessment of cognitive function. The MWM was used to investigate the acquisition of reference and working memory. In addition, the MWM tank was also used to examine spatial ability using a delayed matching-to-sample (DMTS) task, a very sensitive measure of spatial working memory. As can be seen in Figure 3A & 3B, the results of the behavioural testing indicated that bAMC lesions produced severe deficits in cognitive function on both the reference and working memory tasks in the MWM. Mg2+ therapy with either the 1 or 2 mmol dose did not significantly facilitate the acquisition of reference memory in the MWM. However, the mean swim latencies for both MgCl2 groups were greatly improved compared to saline in the last 2 blocks of trials. Although Mg2+ therapy did not demonstrate a statistically significant improvement on reference memory performance in the present study, it did improve working memory performance in the MWM. Administration of the 2 mmol dose of MgCl2 significantly reduced the working memory deficit compared to saline treatment on the first 2 days of testing. The 1 mmol dose of MgCl2 also reduced the working memory impairments on the first day of testing and significantly improved working memory performance on day 2 of testing. Severe cognitive impairments were also seen on

the DMTS spatial memory test following injury. The 2 mmol dose of MgCl2 significantly reduced the number of trials needed to reach the criterion of 80% correct choices compared to saline- treated rats, while the 1 mmol dose of MgCl2 did not, although the number of trials was greatly reduced. Thus, Mg2+ therapy was effective in a task and dose-dependent manner in this study. Mg2+ therapy in the traumatically injured brain To examine the ability of Mg2+ therapy following TBI, groups of rats were prepared with a cortical contusion injury (CCI) or sham procedure and then assigned to either MgCl2 (1.0 mmol/kg, i.p.) or saline treatment conditions (Hoane, 2005). Mg2+ therapy was administered 15 min and 24 hr following injury. Rats were then examined for cognitive/spatial performance in the MWM, investigating the acquisition of reference and working memory. Administration of MgCl2 following CCI significantly reduced some of the behavioural impairments observed following injury (see Figure 3C & 3D). The acquisition of reference memory in the MWM was significantly improved compared to saline-treated rats. In contrast, MgCl2 did not improve working memory performance. In a second study, the ability of Mg2+ therapy to improve cognitive/spatial performance following unilateral CCI was examined. Groups of rats were given unilateral CCIs or sham surgeries of the left sensorimotor motor/frontal cortex. One hr following injury, rats were administered 1 mmol /kg MgCl2 or saline. They were then tested for their ability to acquire a reference memory task in the MWM on 4 consecutive days (4 trials/day) starting 11 days after CCI. Their working memory performance was measured on days 16 and 17. It was found that the single 1 mmol/kg dose of MgCl2 effectively facilitated the acquisition of the reference memory task compared to treatment with saline (see Figure 4). In a similar manner, the working memory performance was greatly enhanced following CCI in the Mg2+-treated rats compared to the saline-treated rats. In fact, the working memory performance of the Mg2+- treated rats could not be distinguished from the sham controls on either day of working memory testing (see working memory graph in Figure 4). Mg2+ treatment appeared to have prevented the occurrence of the working memory deficit following unilateral frontal injury. 119

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Figure 3. The effects of a regimen of MgCl2 (1 or 2 mmol/kg) administered following bAMC focal lesions on cognitive performance in the MWM. Plotted are the mean swim latencies ( SEM) during the acquisition phase of a working memory task (A) and the mean number of trials to criterion in the DMTS test (B) is presented. There was a dose and task-dependent effect on recovery. The 2 mmol dose provided the greatest improvement on both measures of working memory (* = p < 0.05 comparing 2 mmol MgCl2 to saline; ^ = p < 0.05 comparing 1 mmol MgCl2 to saline). Adapted from Hoane et al., (2003). The effects of a regimen of MgCl2 (1 mmol/kg) administered 15 min following bilateral CCI of the frontal cortex. Plotted are the mean swim latencies (+SEM) on the acquisition of reference memory (C) and working memory (D) tasks in the MWM. Administration of MgCl2 significantly improved the acquisition of a reference memory task, but not working memory following CCI (* = p < 0.05). Adapted from Hoane et al., (2005). 120

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Figure 4. The effects of MgCl2 (1 mmol/kg) administration 1 hr following unilateral CCI of the sensorimotor/frontal cortex. Plotted are the mean swim latencies ( SEM) on the acquisition of reference memory and working memory tasks in the MWM. Administration of MgCl2 significantly improved the acquisition of a reference memory task and working memory following CCI. Adapted from Hoane (2007). Discussion cognitive performance in a task-dependent manner. Previous studies that have examined the The results of these studies have demonstrated a ability of Mg2+ therapy to improve cognitive 2+ wide range of conditions in which Mg therapy performance following injury have shown mixed results. For example, administration of MgCl2 regulates cognitive function. It was first shown that daily injections of MgCl2 administered 30 following FPI has been shown to improve cognitive outcome by reducing memory loss in mins prior to training on the task worsened the MWM (Smith et al., 1993), but administration acquisition of the reference memory task. It was of MgCl2 failed to improve the acquisition of a also demonstrated that a two-week regimen of dietary Mg2+ deficiency impaired learning on 3 reference memory task in the MWM following injury (Bareyre et al., 1999). Thus, in a similar different cognitive tasks. This finding is especially manner the current series of studies has shown interesting because the animals had been placed similar mixed results. That is, significant effects back onto a normal laboratory diet prior to the were seen in some cases and non-significant assessment phase of the study. effects were seen in others. However, in general 2+ we saw significant improvements in cognitive Mg therapy in several models of cortical function by MgCl2 in each of our studies. The ablation and TBI have demonstrated positive effects on cognitive recovery. However, these discrepant results mainly varied based on dose effects occurred in a task and dose-dependent and task-dependent properties of the studies. manner. Following focal ablation of the bAMC, MgCl2 improved working memory performance From a mechanistic standpoint, Mg2+ therapy has on several measures and slightly improved multiple routes by which it can disrupt the reference memory performance. The CCI studies pathophysiological processes that occur following performed in our laboratory have shown that injury and enhance cognitive recovery. In MgCl2 administration following injury improved addition to offsetting injury-induced Mg2+ 121

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depletion (McIntosh et al., 1988; Vink et al., 1988) and preventing excitotoxic neuronal death (Nowak et al., 1984) mediated by the NMDA receptor, Mg2+ has been shown to have several other effects. For instance, administration of MgSO4 has been shown to limit the generation of injury-induced edema following closed-head injury (Feldman et al., 1996) and it has been more recently shown that MgSO4 (30 mg/kg) reduced aquaporin-4 immunoreactivity, thus contributing to edema reduction following injury (Ghabriel et al., 2006). Administration of MgCl2 has also been shown to reduce the expression of p53 mRNA, a gene associated with the induction of cell death, following lateral FPI (Muir et al., 1999). In this study it was found that 750 mol/kg of MgCl2 reduced the expression of p53 mRNA in the injured cortex compared to saline-treated controls (Muir et al., 1999). High concentrations of Mg2+ (3 mM) have been shown to inhibit lipid peroxidation (Regan et al., 1998). Regardless of the mechanisms of action, the data presented in this review has shown that MgCl2 has strong biological activity, appears to cross the BBB, and can improve cognitive performance following cortical ablation or TBI. Conclusion The studies presented in this book chapter have demonstrated a wide range of activities for Mg2+ therapy in relationship to cognitive function in References
Barbagallo M, Dominguez LJ (2010) Magnesium and aging. Curr Pharm Des 16:832-9. Bardgett ME, Schultheis PJ, McGill DL, Richmond RE, Wagge JR (2005) Magnesium deficiency impairs fear conditioning in mice. Brain Res 1038:100-6. Bardgett ME, Schultheis PJ, Muzny A, Riddle MD, Wagge JR (2007) Magnesium deficiency reduces fear- induced conditional lick suppression in mice. Magnes Res 20:58-65. Bareyre FM, Saatman KE, Helfaer MA, Sinson GP, Weisser JD, Brown AL, McIntosh TK (1999) Alterations in ionized and total blood magnesium after

the rodent. Daily injections of MgCl2 prior to the acquisition of a learning task blocked the acquisition of a reference memory task and dietary deficiency of Mg2+ impaired learning on a number of different tasks. Using the damaged brain as a model to examine the ability of Mg2+ therapy to improve cognitive performance also demonstrated significant advantages with the therapy. Thus, it does appear that Mg2+ status and therapy have significant effects on cognitive performance in the brain and that further research is warranted. In addition, an accumulating body of research suggests that Mg2+ also plays an important role in human cognitive performance. Given the reported rates of magnesium deficiency in humans it is likely that this could impair cognitive performance (Barbagallo and Dominguez, 2010; Elin, 2010). In addition, the most intriguing factor related to Mg2+ therapy for cognitive wellbeing may reside in the use of Mg2+ as a co-therapy with other vital nutrients, which may produce the strongest effects. Acknowledgements A special thanks is given to Alicia Swan for critical reading of an earlier draft of this chapter. Partial support provided by ARRA funds from NINDS grant NS045647-04
experimental traumatic brain injury: Relationship to neurobehavioral outcome and neuroprotective efficacy of magnesium chloride. J Neurochem 73:271- 80. Billard JM (2006) Ageing, hippocampal synaptic activity and magnesium. Magnes Res 19:199-215. Browne KD, Leoni MJ, Iwata A, Chen XH, Smith DH (2004) Acute treatment with MgSO4 attenuates long- term hippocampal tissue loss after brain trauma in the rat. J Neurosci Res 77:878-83. Elin RJ (2010) Assessment of magnesium status for diagnosis and therapy. Magnes Res. 23:194-8.

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Enomoto T, Osugi T, Satoh H, McIntosh TK, Nabeshima T (2005) Pre-Injury magnesium treatment prevents traumatic brain injury-induced hippocampal ERK activation, neuronal loss, and cognitive dysfunction in the radial-arm maze test. J Neurotrauma 22:783-92. Feldman Z, Gurevitch B, Artru AA, Oppenheim A, Shohami E, Reichenthal E, Shapira Y (1996) Effect of magnesium given 1 hour after head trauma on brain edema and neurological outcome. J Neurosurg 85:131- 7. Ghabriel MN, Thomas A, Vink R (2006) Magnesium restores altered aquaporin-4 immunoreactivity following traumatic brain injury to a pre-injury state. Acta Neurochir Suppl 96:402-6. Gmez-Pinilla F (2008) Brain foods: The effects of nutrients on brain function. Nat Rev Neurosci 9:568- 78. Hallack M, Berman RF, Ortemkauf SM, Evans MI, Cotton DB (1992) Peripheral magnesium sulfate enters the brain and increases the threshold for hippocampal seizures in rats. Am J Obstet Gynecol 167, 1605-10. Hoane MR (2004) Magnesium therapy and recovery of function in experimental models of brain injury and neurodegenerative disease. Clin Calcium 14:65-70. Hoane MR (2005) Treatment with magnesium improves reference memory but not working memory while reducing GFAP expression following traumatic brain injury. Restor Neurol Neurosci 23:67-77. Hoane MR (2007) Assessment of cognitive function following magnesium therapy in the traumatically injured brain. Magnes Res 20:229-36. Hoane MR, Barth TM (2001) The behavioral and anatomical effects of MgCl2 therapy in an electrolytic lesion model of cortical injury in the rat. Magnes Res 14:51-63. Hoane MR, Kaufman NA, Vitek MP, McKenna SE (2009) COG1410 improves cognitive performance and reduces cortical neuronal loss in the traumatically injured brain. J Neurotrauma 26:1-10. Hoane MR, Knotts AA, Akstulewicz SL, Aquilano M, Means LW (2003) The behavioral effects of magnesium therapy on recovery of function following bilateral anterior medial cortex lesions in the rat. Brain Res Bull 60:105-14. Hoane MR, Wolyniak J, Akstulewicz SL (2005) Administration of riboflavin improves behavioral outcome and reduces edema formation and GFAP

expression following traumatic brain injury. J Neurotrauma 22:1112-22. Huskisson E, Maggini S, Ruf M (2007) The influence of micronutrients on cognitive function and performance. J Int Med Res 35:1-19. Kaufman NA, Beare JE, Tan AA, Vitek MP, McKenna SE, Hoane MR (2010) COG1410, an apolipoprotein E- based peptide, improves cognitive performance and reduces cortical loss following moderate fluid percussion injury in the rat. Behav Brain Res 214:395- 401. Kesner RP, Dakis M, Bolland BL (1993) Phencyclidine disrupts long- but not short-term memory within a spatial learning task. Psychopharmacology 111:85-90. Lindner MD (1997) Reliability, distribution, and validity of age-related cognitive deficits in the Morris water maze. Neurobiol Learn Mem 68:203-20. Lindner MD, Plone MA, Cain CK, Frydel BR, Francis JM, Emerich DF, Sutton RL (1998) Dissociable long-term cognitive deficits after frontal versus sensorimotor cortical contusions. J Neurotrauma 15:199-216. McIntosh TK, Faden AI, Yamakami I, Vink R (1988) Magnesium deficiency exacerbates and pretreatment improves outcome following traumatic brain injury in 31 rats: P magnetic resonance spectroscopy and behavioral studies. J Neurotrauma 5:17-31. McLamb RL, Williams LR, Nanry KP, Wilson WA, Tilson HA (1990) MK-801 impedes the acquisition of a spatial memory task in rats. Pharmacol Biochem Behav 37:41- 5. Muir JK, Raghupathi R, Emery DL, Bareyre FM, McIntosh TK (1999) Postinjury magnesium treatment attenuates traumatic brain injury-induced cortical induction of p53 mRNA in rats. Exp Neurol 159:584-93. Murray TK, Ridley RM (1997) The effect of dizocilpine (MK-801) on conditional discrimination learning in the rat. Behav Pharmacol 8:383-8. Nowak L, Bregestovski P, Ascher P, Herbert A, Prochiantz A (1984) Magnesium gates glutamate activated channels in mouse central neurones. Nature 307:462-5. Pussinen R, Sirvio J (1999) Effects of D-cycloserine, a positive modulator of N-methyl-D-aspartate receptors, and ST 587, a putative alpha-1 adrenergic agonist, individually and in combination, on the non-delayed and delayed foraging behaviour of rats assessed in the radial arm maze. J Psychopharmacol 13:171-9.

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Quigley A, Tan AA, Hoane MR (2009) The effects of hypertonic saline and nicotinamide on sensorimotor and cognitive function following cortical contusion injury in the rat. Brain Res 1304:138-48. Regan RF, Jasper E, Guo Y, Panter SS (1998) The effect of magnesium on oxidative neuronal injury in vitro. J Neurochem 70:77-85. Saad L, Silva L, Banzato C, Dantas C, Garcia C (2010) Anorexia nervosa and Wernicke-Korsakoff syndrome: a case report. J Med Case Reports 4:1-5. Sen AP, Gulati A (2010) Use of magnesium in traumatic brain injury. Neurotherapeutics 7:91-9. Slutsky I, Abumaria N, Wu LJ, Huang C, Zhang L, Li B, Zhao X, Govindarajan A, Zhao MG, Zhuo M, Tonegawa S, Liu G (2010) Enhancement of learning and memory by elevating brain magnesium. Neuron 65:165-77. Smith DH, Okiyama K, Gennarelli TA, McIntosh TK (1993) Magnesium and ketamine attenuate cognitive dysfunction following experimental brain injury. Neurosci Lett 157:211-4.

Van Den Heuvel C, Vink R (2004) The role of magnesium in traumatic brain injury. Clin Calcium 14:9-14. Vink R, Cook NL, van de Heuvel C (2009) Magnesium in acute and chronic brain injury: an update. Magnes Res 22:158S-62S. Vink R, McIntosh TK, Demediuk P, Weiner MW, Faden 2+ AI (1988) Decline in intracellular free Mg is associated with irreversible tissue injury after brain trauma. J Biol Chem 263:757-61. Vink R, O'Connor CA, Nimmo AJ, Heath DL (2003) Magnesium attenuates persistent functional deficits following diffuse traumatic brain injury in rats. Neurosci Lett 336:41-4. Wang C-T, Li Y, Wang F-J, Shi Y-M, Lee B-T (2008) Correlation between iron, magnesium, potassium and zinc content in adolescent girl's hair and their academic records. Chang Gung Med J 31:358-62.

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Magnesium in headache
Lisa A. Yablon* and Alexander Mauskop
The New York Headache Center, 30 East 76th Street, New York, NY 10021, USA.
* drmauskop@nyheadache.com

Abstract Magnesium's role in migraine pathogenesis is well-described, with deficiencies known to promote cortical spreading depression, alter nociceptive processing and neurotransmitter release, and encourage the hyperaggregation of platelets, all major elements of migraine development. Research on magnesium has found it to be a potentially well-tolerated, safe and inexpensive option for migraine prevention, while it may also be effective as an acute treatment option for headaches including migraines, tension- type headaches and cluster headaches, particularly in certain patient subsets. This chapter will review the various aspects of migraine in which magnesium plays a part, as well as numerous studies on the use of magnesium in both headache prophylaxis and in the acute treatment of headaches, offering recommendations in its use in clinical practice.


Magnesium in the Body Magnesium (Mg), the second most abundant intracellular divalent cation, is a cofactor of many enzymes and is involved in a plethora of cellular functions. It plays a central role in both glucose metabolism and in ATP function. Over 300 enzymes require the presence of magnesium ions for their catalytic action, including all enzymes utilizing or synthesizing ATP, or those that use other nucleotides to synthesize DNA and RNA. ATP exists in cells as a chelate of ATP and a magnesium ion. Because of the important interaction between phosphate and magnesium ions, magnesium ions are essential to the basic nucleic acid chemistry of life, and thus are essential to all cells of all known living organisms. Magnesium is involved in the formation of phospholipids and the insertion of proteins into the phospholipid membrane, and is therefore critical to membrane stabilization (Durlach et al., 1987). It also contributes to contraction of the cytoskeleton at the myoneural junction, playing a vital role in the function of both skeletal, cardiac and other smooth muscles. Magnesium is absorbed in the gastrointestinal tract, via intestinal epithelial channels in the ileum as well as by the renal systems thick ascending limb, distal tubule, and loop of Henle of the nephron (Wagner, 2007). It facilitates calcium absorption via the thick ascending limb, and the absorption of both ions is regulated by the parathyroid hormone (PTH) secreting cells of the parathyroid gland (Bapty et al., 1998). The calcium/magnesium sensing receptor within the parathyroid gland regulates absorption of both ions by detecting their levels in ionized form, and then controlling PTH secretion, thereby maintain- ing calcium homeostasis (Brown et al., 1993). Dietarily, absorption is affected by protein intake as well as phosphate, phytate and fat. Absorbed dietary magnesium is largely excreted through the urine, although most iatrogenically admin- istered oral magnesium is excreted through the faeces. Adult human bodies contain approximately 24 grams of magnesium, with 67% located in the skeleton, 31% intracellularly (20% in skeletal muscle), and only 1-2% extracellularly. Of this amount, one half is ionized, and 25-30% is protein bound. As a result, levels found on routine serum testing, which only reflects that magnesium found in the extracellular space, is not represent- ative of true total body magnesium stores (Moe, 2008). Serum levels are typically 0.71.0 mmol/L or 1.8 2.4 mEq/L. Serum magnesium levels may appear normal even in cases of underlying intracellular deficiency, and true hypomagnesemia is common, possibly due to decreased intake or 125

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absorption, increased loss via the urine or diarrhea, or genetic factors (Henrotte, 1982). Primary and Secondary Hypomagnesemia Familial hypomagnesemia with secondary hypo- calcemia has been studied in various kindreds, and heredity has been found to be X linked in some families, and autosomal recessive in others (Walder et al., 1997). There are currently more than 30 known mutations in the TRPM6 gene that are associated with familial hypomagnesemia and hypocalcemia. Another hereditary form of hypo- magnesemia, tubular hypomagnesemia/hypo- kalemia with hypocalciuria (Gitelman's synd- rome), is hypothesized to be due to two different types of genetic transmission, one autosomal recessive and one autosomal dominant with high phenotype variability (Bettinelli et al., 1995). A population study in Germany found the prevalence of serum hypomagnesemia to be 14.5%, with even higher frequencies in females (Schimatschek and Rempis, 2001). Additionally, chronic disease is associated with hypo- magnesemia, including diabetes, asthma, cardio- vascular disease, sickle cell anaemia, pre- eclampsia and eclampsia (Laires et al., 2004). 10- 20% of hospitalized patients are deficient in magnesium, and up to 65% of patients in intensive care units are hypomagnesemic. Alcoholism is also associated with inadequate magnesium levels, in part due to poor nutrition (Bohmer and Mathiesen, 1982). It has also been implicated in patients with end-stage renal disease suffering from hemodialysis headache (Goksel et al., 2006), and is often seen in conjunction with electrolyte abnormalities including hypokalemia, hyponatremia, hypo- calcemia and hypophosphatemia (Whang et al., 1985). A number of medications such as diuretics, aminoglycosides and digoxin are associated with hypomagnesemia, and patients with refractory hypocalcemia and hypokalemia should be evaluated for hypomagnesemia (Innerarity, 2000). Magnesium Imbalances Clinical symptoms of hypomagnesemia include hallucinations, depression, delirium, lethargy, weakness, paresthesias, tremors, premenstrual syndrome, cold extremities, leg or foot cramps, 126

seizures, ventricular arrhythmias and congestive heart failure (Douban et al., 1996). However, since total body stores are not accurately represented by serum levels, routine blood testing and even erythrocyte Mg concentrations may reveal normal levels, particularly in patients with low free (ionized) magnesium levels. Urinary fractional excretion or the oral magnesium load test can estimate the total body magnesium status. Intravenous magnesium loading tests are likely the most accurate and practical assessment, whereby total excretion of urinary magnesium is calculated over a 24 hour period, following administration of a loading dose; a retention of 20% or more indicates deficiency (Arnaud, 2008). Hypermagnesemia, on the other hand, is a rare condition due to the nephrons rapid response to increased levels. It usually develops only in people with kidney failure who are given magnesium salts or who take drugs that contain magnesium such as laxatives or antacids. Clinical symptoms include nausea, muscle weakness, lethargy, confusion, hypotension and arrhythmias. In mild cases, withdrawing magnesium supple- mentation is often sufficient. In more severe cases, intravenous calcium gluconate and diuretics or dialysis may be required. Magnesium Levels in Migraineurs For many decades, it was postulated that magnesium deficiency played a role in migraine pathogenesis. However, the lack of simple and reliable measures of magnesium levels prevented further research to prove this theory. While low serum, cerebrospinal fluid and cerebral tissue levels of magnesium have been found to be low in patients with migraine (Jain et al., 1985; Ramadan et al., 1989; Schoenen et al., 1991), these results have been inconsistent, with both normal and low levels detected in the same tissues of some patients. The variability of results may be due to the need to measure ionized magnesium as a true reflection of magnesium metabolism, and the development of an ion selective electrode for ionized magnesium in whole blood, serum, plasma and aqueous samples has made an accurate and rapid measurement of ionized magnesium levels possible (Altura et al., 1992).

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A study measuring ionized magnesium levels in 40 patients during an acute migraine attack found that 50% had levels below 0.54 mmol/l (normal adult range 0.54-0.65 mmol/l) (Mauskop et al., 1995), with all subjects having total serum magnesium levels within normal limits. Intravenous administration of 1g of magnesium sulfate was most effective in those with low ionized magnesium, with 86% of patients reporting sustained pain relief over 24 hours in those found to have low serum ionized magnesium, while this was the case in only 16% of patients with normal levels. This finding was extended to patients with various headache types, including migraine without aura, cluster head-ache, chronic migraine and chronic tension type headache (Mauskop et al., 1996), with most patients demonstrating low ionized magnesium levels. In addition, high serum ionized calcium to magnesium ratios were found in all headache types except for in those patients with chronic tension-type headaches. Based on these findings, it has been suggested that tension type headache may possibly be discriminated from chronic migraine based on serum ionized magnesium levels (Mauskop et al., 1994). Migraine Pathogenesis Migraine is the most common form of disabling primary headache that afflicts patients, affecting approximately 12% of Western populations (Lipton et al., 2007). It is clear that there is genetic transmission of the disorder, although specific genes for most forms of migraine have not yet been identified. Additionally, although the exact etiology remains to be defined, current theories centre on hyperexcitability of the cortex and trigeminovascular complex. In migraineurs, headache triggers stimulate the release of neuropeptides from the trigeminovascular neurons, including calcitonin gene-related peptide and substance P. Additionally, stimulation of the trigeminal ganglion increases cerebral blood flow due to the release of vasoactive intestinal peptide by the facial nerve (Goadsby and Macdonald, 1985). The vasodilation is accompanied by mast cell degranulation, blood vessel edema and increased vascular permeability, resulting in meningeal neurogenic inflammation. The trigeminal nerve transmits this information to the brainstem trigeminal nucleus caudalis, and

then on to the thalamic nuclei and the cortex, where the pain is ultimately perceived (Moskowitz, 1984). Other structures, including the periaqueductal gray matter and the locus coeruleus and the dorsal raphe nuclei, modulate pain transmission and therefore its perception (Martin and Behbehani, 2001). Recent research has elucidated the aura phase of migraine, which affects up to 5% of the adult population (Agostini and Aliprandi, 2006). Migraine aura is the presentation of charac- teristic neurological symptoms usually developing prior to the onset of the painful phase of a migraine headache, believed to be due to a phenomenon known as cortical spreading depression (CSD). CSD was originally described by Leao (1944) and is an intense depolarization of neuronal and glial membranes, with alterations in membrane resistance and ion flow. There is subsequent massive release of glutamate and potassium as well as an increase in intracellular sodium and calcium. This results in a strong wave of depolarization that spreads across contiguous neuronal tissue. It can be triggered by depolarization of a small region of brain tissue or by direct application of excitatory amino acids, and activation of the N-methyl-D aspartate (NMDA) receptor can evoke CSD (Gorji et al., 2001). There are characteristic alterations in cerebral blood flow, with an initial brief oligemia followed by a profound hyperemia, and a mild, long-lasting oligemia (Otori et al., 2003). Precisely how the aura phase of a migraine evolves into the painful phase remains unknown, and it has been theorized that it is due to the action of a number of inflammatory proteins including calcitonin gene-related peptide (CGRP), nitric oxide and vasoactive peptide (Goadsby et al., 1990), which feed into the trigeminal nerve and generate pial artery dilation and CSD, and, ultimately, headache. Using phosphorus nuclear magnetic resonance spectroscopy (MRS), low levels of magnesium have been found in the cerebral tissue of some migraineurs both during attacks and interictally (Ramadan et al., 1989). Another study utilized the same technology to assess the brain cytosolic free magnesium concentration and free energy released by the hydrolysis of adenosine triphosphate, an index of cellular bioenergetics in both migraineurs and patients with cluster 127

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headaches (Lodi et al., 2001). Cytosolic free magnesium and the free energy released by the reaction of ATP hydrolysis were significantly reduced in the occipital lobes of patients with all types of migraine as well as in cluster headache patients. The authors of this study took these results to lend support to their hypothesis that the reduction of free magnesium in tissue with mitochondrial dysfunction is due to a bioenergetics deficit, as magnesium is essential for mitochondrial membrane stability and the coupling of oxidative phosphorylation. Phosphorus nuclear MRS demonstrated reduced magnesium in the occipital cortices of patients with hemiplegic migraine, with decreases correlating with the severity of neurological complaints (Boska et al., 2002). Interestingly, this study also found increased magnesium in the brains of patients with migraine without aura, which the authors attributed to a possible decrease in intracellular potassium, which may occur in neuronal tissue prone to hyper-excitability. Various hypotheses abound as to the reason behind the hypomagnesemia observed in migraineurs. Some suggest that during a migraine headache, excessive amounts of magnesium are excreted due to stress. Others propose that stress triggers excretion of magnesium, with secondary hypomagnesemia causing a migraine (Durlach, 1976). During attacks as well as interictally, magnesium levels in both serum and saliva are decreased (Gallai et al., 1992), perhaps an indicator of low cerebral magnesium levels and therefore a decreased threshold for migraine development (Ramadan et al., 1989). Interictal studies on intracellular and serum levels in patients with migraines and tension-type headaches have shown inconsistent results. However a study of migraineurs utilizing the magnesium load test found that after loading with magnesium lactate, there was retention of the magnesium, suggesting a systemic deficiency. Further, interictal levels of erythrocyte magnesium are lower in both adult (Schoenen et al., 1991) and juvenile migraineurs with and without aura (Soriani et al., 1995). In support of these studies, one in which migraineurs with low erythrocyte magnesium levels as well as decreased ionized lymphoycyte magnesium levels were given mineral water containing magnesium over a two-week period. Both erythrocyte and lymphocyte magnesium levels rose (Thomas et 128

al., 2000). These suggest that assays of erythrocyte magnesium may be a useful and easily available tool to assess magnesium deficiency in migraineurs. Magnesium in Migraine Pathogenesis Magnesium is believed to be involved in a number of the aspects of migraine patho- physiology, and deficiency has been linked to cortical spreading depression (Mody et al., 1987), platelet aggregation (Baudouin-Legros et al., 1986), release of substance P (Weglicki and Phillips, 1992), neurotransmitter release (Coan and Collingridge, 1985) and vasoconstriction (Altura and Altura, 1982). NMDA receptors are associated with nociception and the resulting neuroplastic changes in the trigeminal nociceptive neurons, as well as with regulation of cerebral blood flow (Foster and Fagg, 1987). Magnesium ions may block the NMDA receptor, thereby preventing calcium ions from moving intracellularly, and stopping the calcium's effects on neurons and cerebral vasculature (Coan and Collingridge, 1985). Decreased magnesium levels therefore facilitate the NMDA receptor, increasing its effects on CSD, as well as the effect of glutamate on the NMDA receptor. The NMDA receptor has been shown to have a part in both the initiation and spread of cortical depression (Ferrari, 1992). Magnesium has been shown to block CSD induced by glutamate, and CSD is more easily initiated with decreased magnesium levels (Mody et al., 1987). Nitric oxide (NO) plays a role as a synaptic modulator, affecting nociceptive processing (Meller and Gebhart, 1993) in addition to its involvement in the regulation of blood flow both intracranially and extracranially. It augments the NMDA receptor-evoked currents, thereby facilit- ating glutaminergic transmission (Choi and Lipton, 2000), which, as previously discussed, can be inhibited by magnesium. Production of NO can be inhibited by decreased magnesium levels. CGRP, a neuropeptide, is released from activated trigeminal sensory nerves, is involved in the dilation of intracranial blood vessels and may also increase nociceptive transmission in the brain- stem and spinal cord. It is therefore believed to play a central part in the development of

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migraines. A positive correlation has been demonstrated between migraines and serum CGRP levels, and after the pain of a migraine subsides, levels are observed to return to normal (Coderre et al., 1993). It has hence been hypothesized that inhibition of the release of CGRP either centrally or from the trigeminal nerve, may inhibit intracranial vasodilation thereby aborting migraine attacks. CGRP antagonists lack vasoconstrictive properties and have an advantage over current acute migraine treatment, triptans, which are contraindicated in patients with cardiovascular risk factors due to their vasoconstrictive effects. Circulating CGRP levels have been shown to be decreased by the administration of magnesium in patients with primary Raynauds phenomenon (PRP). A study followed CGRP levels in 12 women with PRP and 12 controls before and after the administration of intravenous magnesium sulfate. While there was no significant difference between baseline circulating CGRP in the two groups, following the infusion of magnesium sulfate there was a significant reduction in CGRP levels in the women with PRP only as well as a significant increase in RBC magnesium levels in the women with PRP but not in the control subjects (Myrdal et al., 1994). Serotonin, released from platelets during migraines, promotes cerebral vasoconstriction as well as triggering nausea and vomiting. Cerebral vascular muscle serotonin receptors may develop increased affinity if serum ionized magnesium falls and the ratio of serum ionized calcium to magnesium increases. This may lead to further cerebral vasoconstriction and facilitates the release of serotonin from neuronal storage sites (Altura and Turlapaty, 1982). Pretreatment with magnesium has been shown to reduce serotonin- induced vasoconstriction (Goldstein and Zsoter, 1978). Oral Magnesium Supplementation A double-blind placebo-controlled study was performed in which 24 women with menstrually- related migraine were given supplementation with 360 mg of magnesium pyrrolidone carbox- ylic acid, divided into 3 divided daily doses taken from ovulation to the first day of menstrual flow (Facchinetti et al., 1991). The treatment was well

tolerated, with only one patient dropping out of the study due to side effects (magnesium- induced diarrhoea), and significant reductions were observed in number of days of headache, total pain index and in Menstrual Distress Questionnaire score. A larger double-blind, placebo-controlled, rand- omized study on 81 migraineurs receiving 600 mg of trimagnesium dicitrate taken once daily, showed attack frequency reduction of 41.6% in the magnesium group and 15.8% in controls. Three patients dropped out of the study due to side effects, 18.6% of patients complained of diarrhoea and 4.7% of gastric irritation (Peikert et al., 1996). A third placebo-controlled double-blind trial showed no effect of magnesium on migraine (Pfaffenrath et al., 1996), possibly attributable to the use of poorly-absorbed magnesium salt, as almost half of the treatment group complained of diarrhoea. A recent double-blind placebo-controlled, randomized study investigated the effects of 600 mg of magnesium citrate supplementation per day in patients with migraine without aura. A combination of clinical assessments, visual- evoked potentials (VEPs) and brain single-photon emission computerized tomography (SPECT) were performed to assess neurogenic and vascular mechanisms of action (Koseoglu et al., 2008). Supplementation was associated with significant decrease in migraine attack frequency and severity, as well as decreased P1 amplitude on VEPs, and increased cortical blood flow to the inferolateral temporal, inferofrontal and insular regions seen on SPECT. The authors suggested that magnesium may interfere with both neurogenic and vascular mechanisms of migraine and may hence be an effective prophylactic treatment. Other studies using SPECT have shown conflicting results (Ramadan et al., 1991; Ferrari et al., 1995; Olesen et al., 1982). Women with menstrually-related migraine (MRM) may be particularly prone to developing magnesium deficiency. A prospective study involving 270 women, 61 of whom had MRM, showed that 45% had ionized magnesium deficiency during MRM attacks, 15% during non- menstrual attacks, 14% during menstruation 129

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without migraine and 15% between menstruation without migraine (Mauskop et al., 2002). Serum ionized calcium/magnesium levels were elevated in MRM, with normal ionized calcium levels. Magnesium deficiency has also been found in paediatric migraine, with decreased levels of serum, RBC and mononuclear blood cell magnesium found in paediatric migraineurs with or without aura compared with patients with tension type headache and controls (Soriani et al., 1995; Mazzotta et al., 1999). Magnesium supplementation may be a safe and well- tolerated option for migraine prophylaxis in the prevention of paediatric migraine. However, while a double-blind placebo-controlled, randomized trial of patients aged 3 to 17 years found a statistically significant downward trend in headache frequency in those treated with magnesium oxide, the difference in the slopes of the two lines was not statistically significant (Wang et al., 2003). It could therefore not be determined if oral magnesium was superior to placebo in preventing frequent migraines in children and adolescents. While oral magnesium is generally well tolerated, the most prominent side-effect is diarrhoea. Magnesium toxicity leads to loss of deep tendon reflexes as well as generalized muscle weakness. Severe toxicity can manifest as cardiac muscle weakness, respiratory paralysis and death, and patients with renal disease are at greater risk for developing toxicity. Although the diarrhoea itself may prevent the development of toxicity, patients should be cautioned regarding excessive intake. Intravenous Magnesium Studies examining the use of intravenous magnesium in the treatment of acute migraine have been conflicting. A study on 40 patients with acute migraine attacks showed an 85% correlation between levels of serum ionized magnesium (measured during an attack) and clinical response to 1g of intravenous magnesium sulphate (Mauskop et al., 1995). Although the study was neither double-blinded nor placebo- controlled, both researchers and subjects were blinded to ionized magnesium levels. A further study on various headache types found 1g of 130

magnesium sulphate to provide rapid relief in patients with low serum ionized magnesium levels (Mauskop et al., 1996). A single-blind placebo-controlled, randomized trial involved 30 patients with migraines who were randomized to receive either magnesium sulphate 1g or placebo (Demirkaya et al., 2001). After 30 minutes, patients in the placebo group who had ongoing pain, nausea or vomiting were given magnesium sulphate 1g. Treatment was superior to placebo in terms of both response rate (100% for magnesium sulphate vs 7% for placebo) and pain-free rate (87% for magnesium sulphate vs 0% for placebo) and those treated did not experience headache recurrence within 24 hours. 87% complained of flushing or a burning sensation in the face and neck. A double-blind placebo-controlled, randomized study evaluated the efficacy of magnesium sulphate 1g on the pain and associated symptoms of migraine with and without aura (Bigal et al., 2002). In subjects with migraine without aura, although there was a significant decrease in the intensity of photophobia and phonophobia, no significant differences were observed in pain relief or nausea. However subjects with migraine with aura had significant improvement in pain and all associated symptoms. An emergency room-based double-blind placebo- controlled, randomized study of 44 subjects with acute migraines tested a combination of magnesium sulphate 2g and metoclopramide 20mg versus metoclopramide 20mg alone at 15 minute intervals for up to 3 doses, or until pain relief occurred (Corbo et al., 2001). Pain intensity was recorded using a standard visual analogue scale (VAS). Although both groups experienced more than 50mm improvement in the VAS score, improvement was smaller in the magnesium group, both comparing VAS score improvements and evaluating normal functional status. The authors suggested that magnesium may diminish the efficacy of metoclopramide in decreasing migraine pain. A study involving 22 patients with cluster headaches who were treated with magnesium sulphate 1g found that 41% of patients reported 'meaningful relief' (defined as complete cessation

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of attacks or relief for more than 3 days) after treatment (Mauskop et al., 1995). Conclusion Magnesium is central to numerous physiological functions, and the role it plays in the various aspects of migraine pathogenesis is well described. Although some studies have shown an association between migraines and magnesium deficiency, it is difficult to assess this with routine blood testing and serum magnesium levels are a poor reflection of body stores of the cation. Therefore, treatment should be based on clinical suspicion, with both oral and intravenous magnesium available as simple, safe, inexpensive References
Agostini E, Aliprandi A (2006) The complications of migraine with aura. Neurol Sci 27(Suppl 2): S91-5. Altura BT, Altura BM (1982) The role of magnesium in etiology of strokes and cerebral vasospasm. Magnesium 1:277-91. Altura BM, Turlapaty PDMV (1982) Withdrawal of magnesium enhances coronary atrerial spasm produced by vasoactive agents. Br J Pharmacol 77:649-59. Altura BT, Shirley TL, Young CC, Dell'Ofrano K, Hiti J, Welsh R, Yeh Q, Barbour RL, Altura BM (1994) Characterization of a new ion selective electrode for ionized magnesium in whole blood, plasma, serum and aqueous samples. Scan J Clin Lab Invest 54 (Suppl. 217):21-36. Altura BT, Shirley T, Young CC, Dell-Ofrano K, Handwerker SM, Altura BM (1992) A new method for 2+ the rapid determination of ionized Mg in whole blood, serum and plasma. Meth Find Exp Clin Pharmacol 14:297-304. Arnaud MJ (2008) Update on the assessment of magnesium status. Br J Nutr 99 (Suppl 3):S24S36. Bapty BW, Dai LJ, Ritchie G, Jirik F, Canaff L, Hendy GN, 2+ 2+ Quamme GA (1998) Activation of Mg Ca sensing 2+ inhibits hormone-stimulated Mg uptake in mouse distal convoluted tubule cells. Am J Physiol 275:F353- 60.

and well-tolerated options for the management of migraines. In patients with symptoms suggestive of hypomagnesemia such as pre- menstrual syndrome, cold extremities and foot or leg cramps, we suggest daily magnesium supplementation with 400mg of chelated magnesium, magnesium oxide or slow-release magnesium. While some patients may require doses of up to 1000mg, diarrhoea and abdominal pain may be limiting factors. Intravenous magnesium may be used in patients who are unable to tolerate or absorb oral magnesium or who are non-compliant with daily dosing. It may also be used for the treatment of acute migraines, or as a monthly prophylactic infusion, often administered premenstrually.
Baudouin-Legros N, Dard B, Guicheney P (1986) Hyperactivity of platelets from spontaneously hypertensive rats. Role of external magnesium. Hypertension 8:694-9. Bettinelli A, Bianchetti MG, Borella P, Volpini E, Metta MG, Basilico E, Selicorni A, Bargellini A, Grassi MR (1995) Genetic heterogeneity in tubular hypomagnesemia-hypokalemia with hypocalciuria (Gitelman's syndrome). Kidney Int 47:547-51. Bigal ME, Bordini CA, Tepper SJ, Speciali JG (2002) Intravenous magnesium sulphate in the acute treatment of migraine without aura and migraine with aura. A randomized, double-blind, placebo-controlled study. Cephalalgia 22:345-53. Bohmer T, Mathiesen B (1982) Magnesium deficiency in chronic alcoholic patients uncovered by an intravenous loading test. Scand J Clin Lab Invest 42:633-6. Boska MD, Welch KMA, Barker PB, Nelson JA, Schultz L (2002) Contrasts in cortical magnesium, phospholipid and energy metabolism between migraine syndromes. Neurology 58:1227-33. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC (1993) Cloning and characterization of an extracellular 2+ Ca -sensing receptor from bovine parathyroid. Nature 366:575-80.

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Choi YB, Lipton SA (2000) Redox modulation of the NMDA receptor. Sal Mol Life Sci 57:1535-41. Coan EJ, Collingridge GL (1985) Magnesium ions block an N-methyl-D-aspartate-mediated component of synaptic transmission in rat hippocampus. Neurosci Lett 53:21-6. Coderre TI, Katz J, Faccerino AL, Melzack R (1993) Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 52:259-85. Corbo J, Esses D, Bijur PE, Iannaccone R, Gallagher EJ (2001) Randomized clinical trial of intraveous magnesium sulfate as an adjunctive medication for emergency department treatment of migraine headache. Ann Emerg Med 38:621-7. Demirkaya S, Vural O, Dora B, Topcuoglu MA (2001) Efficacy of intravenous magnesium sulfate in the treatment of acute migraine attacks. Headache 41:171-7. Douban S, Brodsky MA, Whang DD, Whang R (1996) Significance of magnesium in congestive heart failure. Am Heart J Sep 132:664-71. Durlach J (1976) Neurological manifestations of magnesium imbalance. In: Handbook of Clinical Neurology (Vinken PJ, Bruyn GW, eds), Amsterdam, Holland, pp 454-579. Durlach J, Poenaru S Rouhani S, Bara, M, Guiet-Bara, A (1987) The control of central neuronal hyperexcitability in magnesium deficiency. In: Nutrients and Brain Function (Essman WB, ed) Karger, Basel, Switzerland, pp 48-71. Fachianetti F, Sances G, Borella P, Genazzeni AR, Nappi G (1991) Magnesium prophylaxis of menstrual migraine: effects on intracellular magnesium. Headache 31:298-301. Ferrari MD (1992) Biochemistry of migraine. Pathol Biol (Paris) 40:287-92. Ferrari MD, Haan J, Blokland JA, Arndt JW, Minnee P, Zwinderman AH, Pauwels EK, Saxena PR (1995) Cerebral blood flow during migraine attacks without aura and effect of sumatriptan. Arch Neurol 52:135-9. Foster AC, Fagg GE (1987) Neurobiology. Taking apart NMDA receptors. Nature 329:395-96. Gallai V, Sarchielli P, Coata G, Firenze C, Morucci P, Abbritti G (1992) Serum and salivary magnesium levels

in migraine: Results in a group of juvenile patients. Headache 32:132-53. Goadsby PJ, Edvinsson L, Elkman R (1990) Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 28:183-7. Goadsby PJ, Macdonald GJ (1985) Extracranial vasodilatation mediated by VIP (Vasoactive Polypeptide). Brain Res 329:285-8. Goksel BK, Torun D, Karaca S, Karatas M, Tan M, Sezgin N, Benli S, Sezer S, Ozdemir, N (2006) Is low blood magnesium level associated with hemodialysis headache? Headache 46:40-5. Goldstein S, Zsoter TT (1978) The effect of magnesium on the response of smooth muscle to 5- hydroxytryptamine. Br J Pharmacol 62:507-14. Gorji A, Scheller D, Straub H, Tegtmeier F, Kohling R, Hohling JM, Tuxhorn I, Ebner A, Wolf P, Werner Panneck H, Oppel F, Speckmann EJ (2001) Spreading depression in human neocortical slices. Brain Res 906:74-83. Henrotte JG (1982) Genetic regulation of red blood cell magnesium content and major histocompatibility complex. Magnesium 1:69-80. Innerarity S (2000) Hypomagnesemia in acute and chronic illness. Crit Care Nurs 23:1-19. Jain AC, Sethi NC, Balbar PK (1985) A clinical electroencephalographic and trace element study with special reference to zinc, copper and magnesium in serum and cerebrospinal fluid (CSF) in cases of migraine. J Neurol Suppl 232:161. Koseoglu E, Talashoglu A, Gonul AS, Kula M (2008) The effects of magnesium prophylaxis in migraine without aura. Mag Res 21:101-8. Laires MJ, Monteiro CP, Bicho M (2004) Role of cellular magnesium in health and human disease. Front Biosci 1:262-76. Leao AAP (1944) Spreading depression of activity in cerebral cortex. J Neurophysiol 7:159390. Lipton RB, Bigal ME, Diamon M. AMPP Advisory Group (2007) Migraine prevalence, disease burden, and the need for preventative therapy. Neurology 68:343-9.

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Lodi R, Iotti S, Cortelli P, Pierangeli G, Cevoli S, Clementi V, Soriani S, Montagna P, Barbiroli B (2001) Deficient energy metabolism is associated with low free magnesium in the brains of patients with migraine and cluster headache. Brain Res Bull 54:437-41. Martin VT, Behbehani MM (2001) Toward a rational understanding of migraine trigger factors. Med Clin North Am 85:911-41. Mauskop A, Altura BT, Altura BM (2002) Serum ionized magnesium levels and serum ionized calcium/ionized magnesium ratios in women with menstrual migraine. Headache 42:242-8. Mauskop A, Altura BT, Cracco RQ, Altura BM (1994) Chronic daily headache - one disease or two? Diagnostic role of serum ionized magnesium. Cephalalgia 14:24-8. Mauskop A, Altura BT, Cracco RQ, Altura BM (1996) Intravenous magnesium sulfate rapidly alleviates headaches of various types. Headache 36:156-60. Mauskop A, Altura BT, Cracco RQ, Altura BM (1995) Intravenous magnesium sulphate relieves cluster headaches in patients with low serum ionized magnesium levels. Headache 35:597-600. Mauskop A, Altura BT, Cracco RQ, Altura BM (1995) Intravenous magnesium sulphate relieves migraine attacks in patients with low serum ionized magnesium levels: a pilot study. Clin Sci 89:633-6. Mazzotta G, Strachielli P, Alberti A, Gallai V (1999) 2+ Intracellular Mg concentration and electromyographical ischemic test in juvenile headache. Cephalalgia 19:802-9. Meller ST, Gebhart GF (1993) Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 52:127- 36. Mody I, Lambert JD, Heinemann U (1987) Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. J Neurophysiol 57:869-88. Moe SM (2008) Disorders involving calcium, phosphorous and magnesium. Prim Care 35:215-37. Moskowitz MA (1984) The neurobiology of vascular head pain. Ann Neurol 16:157-68. Myrdal U, Leppert J, Edvinsson L, Ekman R, Hedner T, Nilsson H, Ringqvist I (1994) Magnesium sulphate infusion decreases circulating calcitonin gene-related

peptide (CGRP) in women with primary Raynaud's phenomenon. Clin Physiol 14:539-46. Olesen J, Laurtizen M, Tfelt-Hansen P, Herriksen L, Larsen B (1982) Spreading cerebral oligemia in classical and normal cerebral blood flow in common migraine. Headache 22:242-8. Otori T, Greenberg JH and Welsh FA (2003) Cortical spreading depression causes a long-lasting decrease in cerebral blood flow and induces tolerance to permanent focal ischemia in the rat brain. J Cereb Blood Flow Metab 23:43-50. Peikert A, Wilmzig C, Kohne-Volland R (1996) Prophylaxis of migraine with oral magnesium: results from a prospective, multi-center, placebo-controlled and double-blind randomized study. Cephalalgia 16:257-63. Pfaffenrath V, Wessley P, Meyer C, Isler HR, Evers S, Grotemeyer KH, Tanieri Z, Soyka D, Gobel H, Fisher M (1996) Magnesium in the prophylaxis of migraine: a double-bind, placebo-controlled study. Cephalalgia 16:436-40. Ramadan NM, Halvorsan H, Vande-Linde A, Levine SR, Helpern JA, Welch KMA (1989) Low brain magnesium in migraine. Headache 29:590-3. Ramadan NM, Levine SR, Welch KMA (1991) Interictal cerebral blood flow asymmetries in migraine with aura. Cephalalgia 11(Suppl):42-3. Schimatschek HF, Rempis R (2001) Prevalence of hypomagnesemia in an unselected German population of 16,000 individuals. Magnes Res 14:283-90. Schoenen J, Sianard-Gainko J, Lenaerts M (1991) Blood magnesium levels in migraine. Cephalalgia 11:97-9. Soriani S, Arnaldi C, De Carlo L, Arcudi D, Mazzotta D, Battistella P, Sartori S, Abbasciano V (1995) Serum and red blood cell magnesium levels in juvenile migraine patients. Headache 35:14-6. Thomas J, Millot JM, Sebille S, Delabroise AM, Thomas E, Manfait M, Arnaud MJ (2000) Free and total magnesium in lymphocytes of migraine patients - effects of magnesium-rich mineral water intake. Clin Chim Ecta 295:64-75. Wagner CA (2007) Disorders of renal magnesium handling explain renal magnesium transport. J Nephrol 20:507-10.

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Walder RY, Shalev H, Brennan TMH, Carmi R, Elbeduor K, Scott DA, Hanauer A, Mark AL, Patil S, Stone EM, Sheffield VC (1997) Familial Hypomagnesemia Maps to Chromosome 9q, not to the X Chromosome: Genetic Linkage Mapping and Analysis of a Balanced Translocation Breakpoint. Hum Mol Genet 6:1491-7. Wang F, Van den Eeden SK, Ackerson LM, Salk SE, Reince RH, Elin RJ (2003). Oral magnesium oxide prophylaxis of frequent migrainous headache in children: a randomized, double-blind, placebo- controlled trial. Headache 43:601-10.

Weglicki WB, Phillips TM (1992) Pathobiology of magnesium deficiency: a cytokine/neurogenic inflammation hypothesis. Am J Physiol 263:R734-7. Whang R, Oei TO, Watanabe A (1985) Frequency of hypomagnesemia in hospitalized patients receiving digitalis. Arch Intern Med 145:655-6.

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The role of magnesium in edema and blood brain barrier disruption


Mehmet Kaya*and Bulent Ahishali
Departments of Physiology and of Histology and Embryology, Istanbul Faculty of Medicine, Istanbul University, Capa 34093 Istanbul, Turkey.
* mehkaya@istanbul.edu.tr

Abstract The blood-brain barrier (BBB) is constituted primarily of brain capillary endothelial cells and is a pre- requisite for the maintenance of brain homeostasis that is essential for optimal brain function. However, a variety of pathological conditions, such as sepsis, multiple sclerosis and epilepsia disrupt the BBB integrity and lead to the development of brain edema. Ionized magnesium (Mg2+) is a crucial cofactor that plays an essential role within the cell and regulates a variety of biochemical reactions. Changes in intra- and extracellular Mg2+ concentrations influence the functions of cells and tissues. A growing body of evidence suggests that Mg2+ plays a pivotal role in ameliorating BBB disruption via a number of mechanisms during certain neurological diseases. Systemic delivery of Mg2+ may constitute an alternative approach in the future, both to improve BBB integrity and to decrease brain edema in the course of a variety of diseases involving brain tissue.

Introduction Blood-Brain Barrier The blood-brain barrier (BBB) is composed mainly of brain capillary endothelial cells and represents a dynamic structure that regulates the trafficking of molecules between blood and brain tissue. The passage of many circulating substances from the capillary bed into the brain parenchyma is tightly controlled by physical and enzymatic barriers provided by the endothelial cells of capillaries in the brain parenchyma (Abbott et al., 2010; Cardoso et al., 2010). In this way, the BBB is equipped with regulating means that enable the maintenance of neuronal homeostasis. In addition, the BBB harbours transport mechanisms that provide bidirectional control of exchange of nutrients, electrolytes and neurotoxins, and thus establishes an optimum milieu that is strictly essential to neuronal survival. Although the BBB appears to possess a static structure, it has the ability to adapt readily to sudden changes. About 95% of the microvessels in the brain display BBB properties, and almost every neuron is estimated to be nourished by a distinct capillary vessel (Pardridge, 2005). When regulating its own activities, the mature BBB receives support from at least three different cell types: 1) pericytes which share the same basement membrane with endothelial cells; 2) astrocytes which envelope

99% of the abluminal face of endothelial cells; and 3) neurons (Guo and Lo, 2009; Correale and Villa, 2009). Nevertheless, under normal conditions, BBB function is regulated primarily by capillary endothelial cells (Fisher, 2009; Abbott et al., 2010). In brain tissue, the barrier-type endothelial cells have a continuous basal membrane and do not exhibit fenestrations. These cells contain many mitochondria, but harbour very few caveola (pinocytotic vesicles) in their luminal surfaces. Tight junctions (TJs) between adjacent brain capillary endothelial cells possess occludin and claudin proteins that serve to preserve junctional integrity. Meanwhile, zonula occludens (ZO)-1 and ZO-2, cingulin, cadherin, cathenin, vinculin and actin constitute accessory proteins that aid in the assembly of TJs. Passive diffusion across the BBB mainly depends on the lipid solubility and molecular weight of molecules. Lipophilic substances with molecular weights less than 400-600 Da can pass readily into the brain tissue by passive diffusion. In the normally functioning BBB, oxygen, carbon dioxide, nicotine, ethanol, lipid-soluble substances such as heroin, and amphiphilic drugs (containing both hydrophobic and hydrophilic moieties) are carried by this route. In addition, a number of molecules are transported across the 135

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BBB by other mechanisms including carrier- and receptor-mediated transport, adsorptive-mediated endocytosis and fluid phase-mediated endo- cytosis (Loscher and Potschka, 2005; Pardridge, 2007). However, many pathological conditions alter the functional and structural characteristics of the BBB impairing the maintenance of neural homeostasis. Magnesium Physiology Magnesium ion (Mg2+), the most abundant divalent cation in living cells, exists not only in the intracellular compartment but also in circulation and cerebrospinal fluid (CSF). Serum Mg2+ concentration normally ranges between 1.7-2.3 mEq/L in humans and may decrease during several pathological conditions (Romani and Scarpa, 2000; Musso, 2009). Cell membranes have a particularly low permeability to Mg2+ and hence the ion moves slowly between intracellular and interstitial compartments. Under physiol- ogical conditions, the Mg2+ concentration within the cell is maintained in a relatively narrow range between 0.5 and 1 mM (Dai and Quamme, 1991; Romani and Scarpa, 2000). Mg2+ acts as a regulatory cation at the systemic and cellular levels, and participates in almost all anabolic and catabolic processes in the body. It plays a fundamental role in a wide range of cellular events, biochemical reactions and physiological functions, by activating over 325 enzyme systems, including those involved in ATP synthesis, carbohydrate metabolism, K+ and Ca2+ transport, cell proliferation and membrane stability and function (Grubbs and Maguire, 1987; Saris et al., 2000; Wolf and Trapani, 2008; Barbagallo and Dominguez, 2010). During normal physiological processes, Mg2+ works as a voltage dependent antagonist and a noncompetitive inhibitor of the N-methyl-D- aspartic acid (NMDA) receptors and ion channels in the brain. Although pharmacokinetic and pharmacodynamic studies in rats have shown Mg2+ entry into the brain upon systemic treatment (Hallak et al., 1992; Hoane, 2007), no concomitant rise of Mg2+ in CSF was noted following parenteral administration in humans with brain insults (McKee et al., 2005). A reduction of Mg2+ level within the cell in pathological states can be considered as an injury factor in the brain and may lead to serious 136

biological and metabolic dysfunction. Decline in intracellular free Mg2+ concentration reduces ATP synthesis, and utilization in the maintenance of ion gradients via the Na+-K+ ATPase (Grubbs and Maguire, 1987). Also, the reduction in Mg2+ concentration within the cell results in an impairment of membrane stability by promoting free radical production (Ebel and Gunther, 1980; Bara and Guiet-Bara, 1984). Among the other events associated with Mg2+ deficiency in brain are opening of Ca2+ channels, cellular entry of Ca2+, release of certain neurotransmitters, activ- ation of NMDA receptors, membrane oxidation and activation of nuclear factor kappa B (NFKB) (Weglicki et al., 1994; Altura et al., 2003; Billard, 2006; Rayssiguier et al., 2010). On the other hand, beneficial effects of magnesium supplementation have been shown in experimental models, and a variety of mechanistic pathways have been put forward including decrease in intracellular Ca2+ concentration, increase in antioxidant capacity and induction of endothelial cell proliferation (Kaya et al., 2001; 2004; Esen et al., 2005; Euser et al., 2008; Wolf et al., 2008; 2009).


Brain edema and magnesium Two major types of brain edema, termed cytotoxic and vasogenic edema, were described by Klatzo in the late 1960s, and later two more types were added to the classification (Klatzo, 1967; Marmarou, 2004; Nag et al., 2009). Cytotoxic brain edema is characterized by sustained intracellular water accumulation, causing cellular injury in the absence of BBB damage and involving mainly astrocytes. On the other hand, vasogenic edema results in extracellular water accumulation in brain parenchyma through BBB disruption. The other types of edema are interstitial edema, which is observed in patients with hydrocephalus, and osmotic edema caused by imbalances of osmotically active substances, promoting water influx into cells. Energy depletion followed by a failure of the Na+- K+ ATPase plays a major role in the pathogenesis of cytotoxic brain edema, and increased uptake of Na+ into the cell cannot be equilibrated by the defective pump. Under certain pathological conditions, such as traumatic brain injury (TBI), cerebral ischemia and acute hypertension, the

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brain edema, which is initially cytotoxic, acquires a vasogenic character in the following stages. Brain edema caused by trauma has also been proposed to be mainly vasogenic in nature due to the opening of TJs in the BBB (Unterberg et al., 2004). Magnesium supplementation has been reported to decrease regional brain tissue water content and attenuate brain edema formation after experimental TBI (Okiyama et al., 1995; Feldman et al., 1996). In addition, magnesium treatment protects the blood-spinal cord barrier, improves clinical recovery, and preserves normal spinal cord ultrastructure in experimental spinal cord injury in rats (Kaptanoglu et al., 2003). Aquaporin (AQP)-4, a bidirectional transmembrane water channel expressed mainly in astrocytes and to a lesser extent in barrier type of endothelial cells and pial membranes, may play a crucial role in the pathogenesis of cytotoxic and vasogenic brain edema and aggravation/resolution of ischemic and traumatic brain edema (Amiry- Moghaddam et al., 2003; Zador et al., 2009). Experimental studies focusing on the treatment of brain edema showed beneficial effects of magnesium administered in combination with various pharmacological drugs in animal models (Royo et al., 2003; Sen and Gulati, 2010). Upreg- ulation of AQP-4 in brain injury leads to an increase in brain water content, resulting in brain edema (Taniguchi et al., 2000; Papadopoulos and Verkman, 2005) and treatment with magnesium causes the down-regulation of AQP-4 (Ghabriel et al., 2006) and thereby attenuates brain edema (Okiyama et al., 1995). It has been reported that Mg2+ exerts neuroprotective effects in an anoxic insult by improving the recovery of synaptic transmission and blocking the loss of protein kinase C (PKC) (Libien et al., 2005). These data are mechanistically consistent with the observation that the treatment of the astrocytes with a PKC activator caused a rapid decrease in AQP-4 mRNA and that this effect was inhibited by a specific PKC inhibitor (Nakahama et al., 1999). Among the other mechanisms put forward for the beneficial effects of Mg2+ in decreasing brain edema are restriction of the opening of paracellular pathways through Ca2+ antagonism, alleviation of the oxidative stress, and prevention of hypertensive encephalopathy through reduction in cerebral perfusion pressure (Belfort et al.,

2008; Euser and Cipolla 2009). Mg2+ has also been shown to reduce brain edema and protect brain morphology in experimental cold-injury by inhibition of lipid peroxidation (Turkoglu et al., 2008). Blood-brain barrier and magnesium Mg2+ is slowly transported across the BBB into the brain by transporters and exchangers located in endothelial cell membranes, including the Na+/Mg2+ exchanger, the Mg2+/Ca2+ exchanger and cation channels. Following systemic administration, regional increases in Mg2+ has been detected in the cerebral cortex and hippocampus in rats with an intact BBB (Hallak et al., 1992; Touyz, 2008). In both physiological and pathological conditions, Mg2+ can directly influence BBB properties. Low Mg2+ concentration in the circulation is associated with increase in endothelial permeability, decrease in vasodilator capacity and an increase in the production of vasoconstrictor substances, cytokines and oxidative products (Touyz, 2003; Maier et al., 2004). In pathological conditions with BBB impairment, Mg2+ passes into the extracellular compartment of the brain in significantly higher concentrations and plays an important role in the pathophysiological processes that follow BBB disruption. An elevation in free Mg2+ concentration in capillary endothelial cells increases the proliferation of endothelial cells, restores the cell's ability to generate and utilize ATP for cellular repair mechanisms, and improves disrupted BBB integrity in a variety of insults. In addition to the stimulatory effect of Mg2+ on endothelial cell migration and proliferation, the observation that high Mg2+ concentration facilitates the re-endothelialization of vascular injuries may also provide new insights into the role of Mg2+ in angiogenesis (Maier et al., 2004). A decrease in Mg2+ level in the microcirculation of the cortical structures causes a rapid and progressive damage to microvessels, leading to focal haemorrhages and brain edema (Altura et al., 1991). Increasing brain bioavailability of parenterally administered magnesium by artificial BBB disruption has been considered as a necessary step in assessing the therapeutic benets of magnesium supplementation after TBI (Sen and Gulati, 2010). Magnesium therapy has been shown to be effective in a variety of animal 137

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models of experimental BBB disruption. Accumulated data indicate that magnesium administration improves functional outcome following BBB disruption and decreases brain edema (Okiyama et al., 1995; Feldman et al., 1996; Kaya et al., 2004; Esen et al., 2005; Euser et al., 2008). Treatment with magnesium and MK- 801 (dizocilpine), a noncompetitive NMDA receptor antagonist, either alone or in combination, can reduce brain edema development and help to restore BBB permeability after experimental diffuse brain injury (Feng et al., 2004; Imer et al., 2009). One of the major mechanisms responsible for the pharmacological action of Mg2+ is blockage of NMDA or alpha-amino-3-hydroxy-5-methyl isoxazole-4-proprionic acid (AMPA) channels/ receptors in cerebral vascular system as well as brain parenchyma (Huang et al., 1994). The observation that blockade of NMDA or AMPA receptors could attenuate BBB disruption in focal cerebral ischemia suggest that ionotropic glutamate receptors are involved, at least partly, in BBB disruption (Liu et al., 2010). Magnesium can modulate hypoxic-ischemic events in the cerebral cortex by blocking the action of local putative excitatory amino acid neurotransmitters

and consequently, high extracellular Mg2+ has been shown to be effective in blocking the pathophysiological mechanisms of rupture and spasm in the brain microvasculature (Huang et al., 1994; Chaon et al., 2006). Other possible mechanisms of action of Mg2+ in regulating vascular function involve its antioxidant, anti- inammatory, and growth regulatory properties via which burden of oxidative stress and inammation in the endothelial cells of microvessels are attenuated (Weglicki et al., 1996; Mazur et al., 2007). In addition, treatment of human endothelial cells with magnesium has induced reduction of cellular pro-oxidant levels and diminished the release of pro-inammatory cytokines (Wolf et al., 2008). Although the effects of magnesium on BBB characteristics and the formation of brain edema in various pathophysiological conditions has not been thoroughly elucidated, the studies mentioned above suggest that magnesium provides protective effects on BBB integrity and reduces brain edema by more than one mechanism (Figure 1). Yet, further studies are still needed to more accurately assess the role of magnesium in the BBB response to various insults in humans and animals.

Figure 1. Effects of magnesium on BBB integrity. 138

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Blood-brain barrier, magnesium and traumatic brain injury BBB disruption commonly occurs shortly after experimental and clinical TBI. Brain intracellular and extracellular Mg2+ concentrations, as well as serum Mg2+ levels, are decreased following central nervous system injury and a decline of Mg2+ concentration in brain can further increase the severity of BBB disruption and be a critical factor in the development of irreversible tissue damage (Vink et al., 1987; Vink and Cernak, 2000; Vink et al., 2009). Reduction in brain intracellular free Mg2+ is also associated with brain intracellular acidosis and a concomitant reduction of brain energy stores (Vink et al., 1988; Altura et al., 1995). Magnesium salts, such as magnesium sulphate (MgSO4) or magnesium chloride were shown to penetrate the BBB and cause enhancement of brain intracellular free Mg2+ concentration following TBI (Heath and Vink, 1999). Accumulated data from animal models indicates that administration of magnesium salts into the circulation or extracellular brain compartments can provide an effective therapy for TBI (Vink and Cernak, 2000; Saatman et al., 2001; Hoane, 2007) by improving BBB integrity and decreasing brain edema (Esen et al., 2003). In contrast to the animal studies mentioned above, McKee and colleagues (McKee et al., 2005) described the functional characteristics of the BBB in patients with TBI by using MgSO4 infusions initiated at an average of 5 days after injury. The authors showed that the increased serum Mg2+ concentrations yielded only a marginal increase of total and ionized Mg2+ in CSF and they concluded that the regulation of magnesium by the constituents of the BBB remains largely intact following brain injuries. Meanwhile, in a double- blinded trial conducted to check the validity of animal data in humans, and to explore whether magnesium infusion initiated within 8 hours of major head injury and continued for 5 days would decrease mortality and improve the functional outcome in head-injured patients, it was reported that there was no clinical suggestion of a beneficial effect of the magnesium regimen in these patients (Temkin et al., 2007). Based on the above-mentioned data, it can be concluded that although there has not been a

consensus between animal and human studies regarding the efficiency of magnesium administration in TBI, it can be effective in the recovery of BBB damage at least in animal models. Blood-brain barrier, magnesium and seizures Eclampsia is a serious hypertensive disorder of pregnancy with seizures and associated BBB disruption and vasogenic edema in a similar manner to that observed in hypertensive encephalopathy (Schwartz et al., 2000; Euser and Cipolla, 2009). In animal models, magnesium treatment has been shown to contribute to the protection of the BBB during eclampsia, to decrease the increased BBB permeability, and to prevent the development of brain edema in certain experimental settings, including acute hypertension and hypoglycemia- induced seizures (Kaya et al., 2001 and 2004; Euser et al., 2008). The above-mentioned beneficial effects of magnesium on BBB integrity may be related to its ability to scavenge free radicals. The antioxidant action of magnesium has been reported in two recent studies (Ariza et al., 2005; Turkoglu et al., 2008), suggesting that it could protect against free radical surge associated with epileptic seizures. However, the exact mechanism/s of action of magnesium treatment in the improvement of BBB integrity in eclampsia still remains to be elucidated. Blood-brain barrier, magnesium and sepsis Magnesium supplementation is one of the experimental methods and pharmacological approaches developed for the treatment of BBB disruption and brain edema caused by septic encephalopathy. The impairment of BBB integrity during sepsis has been shown in several studies, and magnesium administration in the early stages of sepsis reduced BBB permeability and brain edema (Papadopoulos et al., 2005; Esen et al., 2005). Magnesium deficiency leads to an elevation in plasma inflammatory cytokines and excessive production of free radicals, and aggravates endotoxic shock (Weglicki et al., 1994; Matsui et al., 2007). Treatment with magnesium decreases the concentration of inflammatory cytokines and free radicals and increases antioxidant capacity and survival rate in rats (Salem et al., 1995). Finally, alterations in endothelial cells, which are protagonists in the 139

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vascular changes during inflammation, are reversible upon magnesium supplementation (Mazur et al., 2007). Although the above- mentioned studies suggest that magnesium is involved in the protection of the BBB and brain edema in sepsis, the literature data is quite limited at present and additional studies are needed to explain the pathophysiologic mechanisms involved in this protection. Blood-brain barrier, magnesium and brain hypoxia/ischemia Cerebral hypoxia/ischemia is known to cause disruption of BBB integrity, thereby increasing the permeability of the BBB and leading to the development of brain edema. Extracellular Mg2+ concentration has been shown to significantly decrease to approximately 60% of basal values in the ipsilateral cortex in hypoxia-ischemia (Lee et al., 2002). Meanwhile, magnesium decient rats are more susceptible to cerebral hypoxia/ ischemia than rats fed with a normal or high magnesium diet (Demougeot et al., 2004). Magnesium administration significantly attenuates the hypoxia-induced increase in reactive oxygen species and contributes to the repair of the disrupted BBB in hypoxia/ischemia (Ravishankar et al., 2001; Goi-de-Cerio et al., 2009). The protection of the BBB by magnesium in hypoxic conditions could be multifactorial and, in addition to the above mentioned effects, may involve other factors such as decrease in the production of cytokines, increase in antioxidative products and blockade of NMDA or AMPA channels/ receptors. There is at present only limited knowledge about the role of magnesium on BBB integrity and brain edema in hypoxia/ischemia and future research is needed to determine the possible mechanisms of magnesium supplement- ation in improving the functions of BBB. Blood-brain barrier, magnesium and ethanol Acute and chronic ethanol treatment gives rise to significant increases in BBB permeability to a variety of molecules that do not normally cross the BBB. Increase in free radical concentration in ethanol-treated endothelial cells leads to phosphorylation of TJ proteins, activation of paracellular pathway and thus disruption of the BBB (Haorah et al., 2005). A number of studies have provided evidence for the alterations in 140

serum Mg2+ levels in acute and chronic ethanol treatments. A single dose of ethanol (2 g/kg) injected to mice significantly decreased total Mg2+ concentration in serum (Papierkowski et al., 1998). Furthermore, acute or chronic alcohol consumption impairs Mg2+ transport and homeostasis at the capillary level in the brain (Romani, 2008). However, little is known about whether any improvement in BBB integrity can be achieved by magnesium treatment in acute or chronic ethanol intake. Therefore, this lack of basic knowledge compounds the difficulty we face when interpreting the importance of efforts of increasing serum Mg2+ levels under these conditions. Blood-brain barrier, magnesium and hydrocephalus Human and animal studies have indicated that hydrocephalus leads to disruption of the BBB. In a study involving a total of 21 patients with normal- pressure hydrocephalus, a slight plasma-like protein pattern has been demonstrated in CSF in 38% of the patients prior to surgical intervention, indicating BBB dysfunction (Wikkels and Blomstrand, 1982). There is only one study in the current literature that evaluates the effects of magnesium in hydrocephalus, and a mild protection against brain damage was shown using MgSO4 therapy in a rat model of childhood-onset hydrocephalus (Khan et al., 2003). Further studies are necessary to increase our understanding of the effects of deficiency or supplementation of magnesium on the functional and structural characteristics of the BBB during hydrocephalus in both animal and human studies. Conclusion It is clear from the discussion above that magnesium plays a variety of essential roles within the cell by modulating the activity of more than 325 enzymes as a cofactor. These enzymes are important for the survival of various cell types including endothelial cells of the BBB. Meanwhile, magnesium deficiency leads to or worsens a variety of central nervous system pathologies by increasing inflammatory cytokines and reactive oxygen species and disturbing the activity of transporters in neurons, astrocytes, pericytes and capillary endothelial cells, which together constitute the neurovascular unit of the brain.

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Besides, the alterations in the Mg2+ concentration in intra- and extracellular fluids are associated with development or aggravation of BBB disruption and brain edema in various clinical disorders and experimental settings. On the other hand, magnesium supplementation can play multiple roles in protecting BBB integrity and improving brain edema. However, owing to the availability of limited knowledge, it is hard to

come to a full understanding of the highly specific actions of magnesium on BBB integrity and brain edema in the course of a variety of pathophysiologies involving the BBB. For this reason, research in this field should continue in order to provide a thorough explanation of the impact of magnesium on the BBB, brain edema and related pathologies.


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Liu X, Hunter C, Weiss HR, Chi OZ (2010) Effects of blockade of ionotropic glutamate receptors on blood- brain barrier disruption in focal cerebral ischemia. Neurol Sci 31:699-703. Lscher W, Potschka H (2005) Blood-brain barrier active efflux transporters: ATP-binding cassette gene family. NeuroRx 2:86-98. Maier JA, Bernardini D, Rayssiguier Y, Mazur A (2004) High concentrations of magnesiummodulate vascular endothelial cell behaviour in vitro. Biochim Biophys Acta 1689:6-12. Marmarou A (2004) The pathophysiology of brain edema and elevated intracranial pressure. Cleveland Clin J Med 71:S6-8. Matsui T, Kobayashi H, Hirai S, Kawachi H, Yano H (2007) Magnesium deficiency stimulated mRNA expression of tumor necrosis factor-a in skeletal muscle of rats. Nutrition Research 27: 66-8. Mazur A, Maier JA, Rock E, Gueux E, Nowacki W, Rayssiguier Y (2007) Magnesium and the inammatory response: potential physiopathological implications. Arch Biochem Biophys 458:48-56. McKee JA, Brewer RP, Macy GE, Phillips-Bute B, Campbell KA, Borel CO, Reynolds JD, Warner DS (2005) Analysis of the brain bioavailability of peripherally administered magnesium sulfate: a study in humans with acute brain injury undergoing prolonged induced hypermagnesemia. Crit Care Med 33:661-6. Musso CG (2009) Magnesium metabolism in health and disease. Int Urol Nephrol 41:357-62. Nag S, Manias JL, Stewart DJ (2009) Pathology and new players in the pathogenesis of brain edema. Acta Neuropathol 118(2):197-217. Nakahama K, Nagano M, Fujioka A, Shinoda K, Sasaki H (1999) Effect of TPA on aquaporin 4 mRNA expression in cultured rat astrocytes. Glia 25:240-6. Okiyama K, Smith DH, Gennarelli TA, Simon RP, Leach M, McIntosh TK. (1995) The sodium channel blocker and glutamate release inhibitor BW1003C87 and magnesium attenuate regional cerebral edema following experimental brain injury in the rat. J Neurochem 64:802-9. Papadopoulos MC, Verkman AS (2005) Aquaporin-4 gene disruption in mice reduces brain swelling and mortality in pneumococcal meningitis. J Biol Chem 280:13906-12.

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Magnesium and hearing loss


Isabelle Sendowski,1,* Xavier Holy,2 Florent Raffin 1 and Yves Cazals 3
1

Institut de Recherche Biomdicale des Armes, Antenne de la Tronche, La Tronche, France. Institut de Recherche Biomdicale des Armes, Antenne de Brtigny, Brtigny-sur-Orge, France. 3 UMR6231, Universit Paul Czanne, Marseille, France.
2

* Isendowski@crssa.net


Abstract Hearing loss is a major public health problem with a large number of causes. Among them, noise-induced hearing loss, drug ototoxicity and sudden sensorineural hearing loss have been proven to result, in part, from metabolic disorders. Metabolic disorders have multiple origins such as ionic, ischemic, excitotoxic and production of cochlear free radicals causing cell death, via necrosis or apoptosis. The efficacy of magnesium, administered either to prevent or to treat hearing damage, has been demonstrated in several studies in animals and in humans, particularly in noise-induced hearing loss. The exact mechanism by which Mg2+ acts is not fully known. Different hypotheses exist including calcium antagonism, vasodilatation, antioxidant and anti-NMDA properties. Because it is a relatively safe and well-known treatment, magnesium therapy, alone or in association, could be of a great interest to improve auditory recovery.

Introduction According to the World Health Organization, 278 million people worldwide have moderate to profound hearing loss in both ears. Besides pathologies of unknown origin, such as sudden hearing loss and Menieres disease, hearing loss has so many known causes; it would be arduous to list them all. Briefly, it can be due to the aging process, exposure to loud noise, certain medications, infections, head or ear trauma, congenital or hereditary factors, diseases, as well as a number of other causes. In noise-induced hearing loss (NIHL), sudden sensorineural hearing loss (SSHL) or iatrogenic hearing loss (ototoxicity), sensory cell death involves metabolic processes. Pharmacological intervention to prevent or ameliorate the evolution of these hearing impairments, as well as susceptibility factors, have been studied for a number of years in animal models or in humans. Among them, magnesium seems to play an important role. Magnesium therapy is well documented because it is usually prescribed in other pathologies. Its side effects and contraindications are few and it is cheap. Magnesium, which easily crosses the hematocochlear barrier, presents neuro- protective and vasodilatory effects, and is able to limit cochlear damage. These observations have led to many investigations, the aim of them being

to evaluate the pertinence of magnesium administration in prevention or treatment in such a hearing impairment. This article presents some arguments that emphasize the interest of magnesium therapy in some forms of hearing loss. How can we hear? Sound waves are transmitted via the bones of the middle ear to the fluid environment of the inner ear, where the sensory organ is in the cochlea. The human cochlea is a 30-35 mm long coiled tube containing three parallel chambers (Figure 1A): the scala vestibuli and the scala tympani, which both contain perilymph, and the scala media, which contains endolymph. In contrast to perilymph, which is similar to cerebrospinal fluid, endolymph contains a large amount of potassium (154 mM) maintained by the cells of the stria vascularis. The organ of Corti (Figure 1B) is composed of supporting cells and hair cells. Of all the sensory organs, the organ of Corti functions with the smallest number of sensory receptor cells. The human cochlea only contains about 15,000 hair cells, including approximately 3,000 inner hair cells (IHCs) and 12,000 outer hair cells (OHCs). The IHCs form one row (Figure 2A) and are the primary sensory receptors. OHCs are organized into three (and sometimes four) rows 145

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Figure 1. Schematic representation of the inner ear. A: section of the cochlea; B: organ of Corti. along the outer edge of the organ of Corti. The longer stereocilia of the OHCs are connected to the tectorial membrane. In the hearing process, sound reaches the inner ear and the basilar membrane supporting the hair cells is displaced. When the hair bundle is deflected, transduction channels are opened and K+ enters the hair cells. The depolarisation evoked by this transduction current activates voltage- gated Ca2+channels and Ca2+ influx. In IHCs, increased intracellular Ca2+ causes mobilization of synaptic vesicles and exocytotic release of glutamate at the base of the hair cells. Glutamate release modulates the activity of the auditory nerve fibres by activating specific receptors, among them the glutamate ionotropic receptors (AMPA, NMDA and Kainite) and the metabo- tropic receptors. The OHCs respond quite differently to changes in membrane potential. Their membranes include a protein (prestin), which alters conformation with the membrane potential and forces cell length changes at acoustic frequencies. This mechanism is thought to amplify and tune the mechanical responses of the basilar membrane. This sensory system is so efficient that, near auditory threshold, a sound vibration of less than 0.1 nanometres is detected, corresponding to a stereocilia displacement 10,000 times lower than its diameter (about one 146

micrometre). However, exposure to loud noise can weaken the system. Magnesium and noise-induced hearing loss (NIHL) Excessive noise is the predominant cause of permanent sensorineural hearing loss. Reports from WHO (2004) state that worldwide, 16% of the disabling hearing loss in adults is attributed to occupational noise, ranging from 7% to 21% in the various subregions. At least 30 million people in the United States encounter hazardous levels of noise at work, particularly in jobs such as construction, mining, agriculture, manufacturing, transportation and in the military. Moreover, the incidence of NIHL continues to grow, partly due to the growing popularity of portable music players with highly efficient headphones (Zhao et al., 2010). The hearing loss can be caused by a single exposure to very loud sounds (impulse noise) or by repeated exposure to louder sounds over an extended period. It may be temporary (temporary threshold shifts, TTS) or permanent (permanent threshold shifts, PTS). It is frequently associated with tinnitus. The mechanisms of damage are of dual origin, mechanical and metabolic. Mechanical damage is immediately developed when the movements of the basilar membrane are excessive, thus inducing detachment of the hairs of the tectorial membrane, disconnection of the interciliary bridges (Figure 2D), or even rupture of membranes. Modern research has provided new insights into the biological mechanisms of NIHL such as free radical production (oxidative stress), glutamatergic excitotoxicity, and ionic and ischemic disorders. They are responsible for delayed hair cell death by necrosis and apoptosis. Oxidative stress It has been demonstrated that an increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) is involved in noise trauma (see Henderson et al., 2006 for review). The superoxide radical anion, nitric oxide (NO) and its redox-related forms, in conjunction with an imbalance of antioxidant defences, have been demonstrated to play a significant role in NIHL as they largely participate in cellular mechanisms that underlie hair cell death. ROS ototoxicity is believed to be associated with deleterious cellular

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Figure 2. A, B: normal aspect of the hair cells and their stereocilia, observed with scanning electron microscopy. C and D: Organ of Corti after traumatizing noise exposure: OHCs are partially missing (C). The stereocilia of hair cells are abnormally bent with broken interciliary bridges (D). intracellular Ca2+ levels. The increase in cytosolic effects at multiple sites, including lipid peroxidation, DNA strand breaks, alteration in Ca2+concentration is not only caused by an influx carbohydrate and protein structures, and a of extracellular Ca2+, but also by the release of triggering of cell death gene expression, leading calcium ions from intracellular stores such as the to necrosis or apoptosis. Oxidants are also endoplasmic reticulum or mitochondria. Dendritic initiators of intracellular cell death signalling swelling and vacuolization are a result of pathways that may lead to apoptosis. The noise- excessive postsynaptic ion influx into the VIIIth induced ROS formation may occur with a delay of nerve terminals at the inner hair cell synapse 7-10 days following exposure to noise (Yamashita (Pujol et al., 1990). et al., 2004). Ionic disorders Excitotoxicity High-level noise stimulation results in a massive Excitotoxicity is a phenomenon of biochemical entrance of potassium through the apical events, triggered by the interaction of excitatory channels of the stereocilia. Moreover, mechanical amino acids with ion channel-bound receptor damage with ruptures of the membranes lining complexes, that can lead to cell death. During the endolymphatic spaces, could involve an high-level noise exposure, the IHCs are highly excessive influx of K+. This increased K+ active, leading to the release of large amounts of concentration may be toxic for the hair cells glutamate into the synapses with the auditory (Zenner, 1986). The consecutive increase in fibres. The level of glutamate in the synapses can intracellular Ca2+ over-activates a series of overstimulate the glutamate receptors, especially enzymes including phospholipases, proteases, the NMDA receptors, and result in high and endonucleases. The result is membrane 147

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breakdown, depolymerization of microtubules and disruption of protein synthesis. Ischemic process Unlike most tissues in which increased metabolism increases blood flow to provide additional oxygen to stressed cells, the cochlea shows reduced blood vessel diameter and red blood cell velocity (Quirk and Seidman, 1995) and decreased blood flow (Thorne and Nuttal, 1987) post-noise. This noise-induced vasoconstriction is a direct consequence of noise-induced formation of 8-isoprostane-F2a, a vasoactive by-product of free radicals (Miller et al., 2003). Cochlear ischemia is aggravated in animals with low Mg2+ content (Scheibe et al., 2000a). These different mechanisms can lead to cell death, which may be more or less rapid, due to necrosis or apoptosis (Yang et al., 2004). Recent studies have revealed that these two types of cell death exist following exposure to intense noise and that apoptosis appears extremely rapidly after the noise stress. Apoptosis has been shown to be the primary cell death pathway in the first day following noise exposure (Hu et al., 2002; 2006). Contrary to birds or reptiles, a mammals auditory hair cells do not regenerate if they are destroyed. Thus, these new metabolic insights bring hope for possible prevention or treatment to limit oxidative stress, ischemia and the apoptosis cascade. A large number of treatments have been tested over the last few years (see Le Prell et al., 2007a; Shibata and Raphael, 2010 for reviews). Among them, magnesium has aroused some interest. Over a number of years, researchers and clinicians have demonstrated the influence of magnesium in the susceptibility to recover following acoustic trauma. NIHL of guinea pigs was found to increase with decreasing Mg2+ content of the drinking water while the Mg2+ content of the food was low and constant. In Mg- deficient guinea pigs, the hearing threshold shift after 10 days of continuous noise exposure was negatively correlated to the Mg2+ content of the perilymph (Ising et al., 1982). Coherent results were obtained in rats (Joachims et al., 1983). Similarly, electrocochleographic measurements of the auditory threshold shifts induced by 148

impulse noises (Devrire et al., 1991) showed that Mg-deficient animals are slightly more susceptible to this type of noise. This susceptibility is less pronounced than after a long duration exposure to a continuous noise, as observed in the previous experiments (Ising et al., 1982). After exposure to continuous noise, up to 75% of the variance of PTS in guinea pigs could be explained by the level of perilymph Mg2+ (Vormann and Gnther, 1993). The increased susceptibility to NIHL with Mg2+ deficit has not only been demonstrated in animal experiments. In a retrospective study in humans, subjective thresholds across frequencies of 3, 4 and 5 kHz were negatively correlated with serum magnesium (Joachims et al., 1987). This finding was the first indication that magnesium status in humans may be one of the factors determining variations in sensitivity to noise-induced hearing loss. Gnther et al., (1989) reported that NIHL observed in 24 air force pilots was negatively correlated to serum Mg2+ concentration. However, Walden et al., (2000), exploring the susceptibility of soldiers to NIHL, failed to demonstrate any correlation between audiometric index and body magnesium. Because Mg2+ deficiency increases the susceptibility to NIHL, several studies have been conducted in animals or in humans to point out the possible prophylactic efficacy of magnesium. Joachims et al., (1983) observed that guinea pigs with physiologically high Mg2+ levels, when exposed to a single shot impulse or a series of impulses, had significantly smaller threshold increases as compared to physiologically low Mg2+ animals. Scheibe et al., (2000b) showed that oral magnesium supplementation significantly reduces TTS and PTS in guinea pigs subjected to a series of impulses. The mean PTS was found to correlate negatively with the total Mg2+ concentration of perilymph and plasma. Conversely, they did not observe any significant effect on PTS following exposure to a gunshot noise. More recently, Attias et al., (2003) explored the activity of the outer hair cells in guinea pigs by means of otoacoustic emission after impulse noise exposure. In animals supplemented with Mg2+, the thresholds were less significantly affected by noise exposure and the audition recovery was faster. In humans, preventive administration of magnesium has also been shown to be effective in noise-related

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hearing loss. Attias et al., (1994) tested the prophylactic effect of magnesium in human subjects exposed to hazardous noise. The study was carried out in 300 young, normal-hearing recruits who underwent 2 months of basic military training. This training included repeated exposure to high levels of impulse noises while using earplugs. The subjects received an additional drink daily containing either 167 mg magnesium aspartate or a placebo. The NIHL was significantly more frequent and more severe in the placebo group (28.5%) than in the magnesium group (11.2%). Moreover, the severity of the NIHL was negatively correlated to the magnesium content of red and mononuclear cells. This prophylactic effect in humans was confirmed by Attias et al., (2004) for temporary threshold shifts. Subjects were exposed to a traumatizing noise over 10 min in order to produce TTS without PTS. Compared to a placebo, the preventive oral intake of magnesium (122 mg Mg2+ aspartate during 10 days) provided significant protection against TTS. A negative correlation between the blood magnesium levels and the TTS was also noted. Whilst magnesium is well known as a preventive treatment for sound trauma, it may also be useful therapeutically. In animals submitted to a series of sound impulses, the systemic administration of magnesium significantly reduced hearing loss after 7 days (Scheibe et al., 2001; 2002). This treatment was more effective if it was quickly instigated. Threshold shift reduction 7 days after acoustic trauma in guinea pigs was confirmed using distortion product otoacoustic emissions (DPOAE, a non invasive technique to test OHC function)(Haupt et al., 2003). In another study (Sendowski et al., 2006) it has been shown that a 7-day post-trauma magnesium treatment reduced auditory threshold shift measured seven days after gunshot noise exposure. However, this improvement was temporary, suggesting that it could be potentially beneficial to prolong the magnesium administration. This hypothesis has since been confirmed (Abaamrane et al., 2009). The latter study compared 4 post-NIHL treatments a 7-day magnesium treatment, a 1- month magnesium treatment, conventional therapy with methylprednisolone, or a placebo. It was demonstrated that 3 months after an impulse noise trauma, the 1-month Mg treatment preserved more hair cells from death

(Figure 3), but this preservation was found to be partial. These observations suggest that post- NIHL treatments can be improved if continued beyond the period of one week. Another track of research concerns combination of different therapies, including magnesium. Treatment with a combination of vitamins A, C, E and magnesium, initiated 1 hour before noise exposure, produced a compelling reduction in NIHL and cell death, while effects of either the antioxidant agents (vitamin A, C, or E) or magnesium alone were small and not statistically reliable (Le Prell et al., 2007b). Thus, the combination of magnesium with other agents could also be of great interest, and suggests that they act at different and complementary levels of the cellular death process. Magnesium and ototoxicity Various pharmaceuticals are known to impair the auditory system. The best-known substances are the aminoglycoside antibiotics and cisplatin. Both cause high frequency sensorineural hearing loss, which is usually permanent and associated with loss of outer hair cells in the basal turn of the cochlea. Aminoglycoside antibiotics (such as gentamicin, neomycin and kanamycin) are used to treat bacteria not responsive to conventional antibiotics. Their clinical use is limited by toxic side effects that include cochlear toxicity, vestibular toxicity and nephrotoxicity. Amino- glycoside antibiotics possibly cause formation of free radicals by forming complexes with iron, which is vital for normal mitochondrial function. Free radicals can rapidly react with cell constituents, including cell membranes and DNA. The resulting oxidative stress can trigger apoptotic cell death. The intrinsic apoptosis pathway is the major pathway induced by aminoglycosides in the cochlea (Rybak and Withworth, 2005). Vormann and Gnther (1991) demonstrated that Mg deficiency in growing rats aggravated the ototoxic effects of the aminoglycoside antibiotic, gentamicin. Administration of gentamicin to Mg2+ deficient pregnant rats from day 16-20 of gestation produced hearing threshold shifts in the maternal rats as well in their offspring (Vormann and Gnther 1991). These data suggest that Mg2+ deficiency is a relevant predisposing 149

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Figure 3. Global hair cells loss in guinea pigs, 3 months after an impulse noise exposure, for 4 treatments groups: placebo (NaCl), a 7-day or 1-month magnesium treatment, and conventional therapy with methylprednisolone. (* p<0.05; **p<0.01) (after Abaamrane et al., 2009). Other well-known ototoxic treatments are risk factor for the development of ototoxicity platinum compounds. Cisplatin is a frequently induced by some pharmaceuticals. Recently, it used chemotherapeutic agent in the treatment of has been shown, in the zebrafish model, that many types of neoplasias, especially of the head extracellular magnesium or calcium ions and neck (Rybak and Withworth, 2005). The modulate hair cell death from neomycin and mechanism of antineoplastic action is associated gentamycin, but exert their protective effects with the selective and persistent inhibition of the through different mechanisms (Coffin et al., deoxyribonucleic acid synthesis (Williams and 2009). In mammals, preliminary injection of Whitehouse, 1979). Its side effects include magnesium solution, by means of ototoxicity, kidney toxicity, medullar suppression electrophoresis, reduced the ototoxic effect of and gastrointestinal disorders. The nephrotoxic kanamicin (Spasov et al., 1999). 150

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effect of cisplatin is severe damage in the renal proximal cells within the thick ascending loop of Henle, manifested by hypomagnesemia. These types of toxicity can impact treatment, as they reduce the chemotherapy dosage, frequency and duration for many patients. Some audiometric studies have reported elevated hearing threshold in 75-100% of patients treated with cisplatin (McKeage, 1995). The auditory lesions seem to result from free radical-induced damage to many tissues (Kopke et al., 1997; Rybak et al., 1995; Dehne et al., 2001). Oxygen reactive species are generated in the cochlea after exposure to cisplatin (Clerici and Yang, 1996) and such oxidative stress can cause cochlear cell death by apoptosis secondary to the activation of caspase-3 (Garcia-Berrocal et al., 2007; De Freitas et al., 2009). Inner ear cell apoptosis can be triggered by the formation of complexes between cisplatin and the DNA of the damaged cell, preventing the progression of the cell cycle. Cevette et al., (2000) observed a significant increase in otoacoustic emission amplitude (which provides frequency-specific information about OHC function) in two patients undergoing cisplatin treatment supplemented with magnesium. The authors of this study concluded that Mg2+ might be a promising agent against cisplatin ototoxicity. However, the study of Sahin et al., (2006) showed that a Mg2+ rich diet prevented the severe hypomagnesemia that cisplatin causes in guinea pigs, but failed to provide any protection against its ototoxic effect. Magnesium and sudden sensorineural hearing loss The US National Institute for Deafness and Communication Disorders defines SSHL as the idiopathic loss of hearing of at least 30 dB over at least 3 contiguous test frequencies occurring within 3 days. Estimates of the overall incidence of SSHL range from 5 to 20 per 100 000 persons per year (Conlin and Parnes, 2007). Many etiologic causes of SSHL have been proposed, but many remain unconfirmed. In the majority of patients (71%), the aetiology remains idiopathic. A wide range of disorders might lead to the development of SSHL. The theorized major causative factors can be broken down into several categories: 1) viral infection (13 %); 2) vascular impairment; 3) immune-mediated

mechanisms; 4) inner ear abnormalities; and 5) CNS abnormalities, including tumours, trauma, haemorrhage, infarction and other pathologies (Chau et al., 2010). The lack of understanding of the mechanism of SSHL has rendered the development of a specific treatment difficult, and currently, empirical guidelines are used. Because of the spontaneous recovery rates of 32% to 70% (Colin and Parnes, 2010), some otolaryngologists choose not to treat SSHL. However the most common approach to treatment of SSHL is with systemic steroids in moderate doses. Nageris et al., (2004) evaluated the efficiency of two treatments after SSHL in humans. The first one consisted of corticosteroid and magnesium, and the second one (control) in corticosteroid and placebo. They observed that more patients treated with magnesium experienced hearing improvement, and at a larger magnitude, than control subjects. Gordin and colleagues (2002) reported a significantly greater recovery rate among patients treated with magnesium and carbogen vs patients treated with carbogen alone. How does magnesium therapy work? Magnesium plays an essential role in the regulation of most cellular functions. However, it is recognized that magnesium status in humans is often deficient. In the recent French SU.VI.MAX study (the supplementation en vitamines et mineraux antioxidants study) of 5,448 subjects, it was shown that 77% of women and 72% of men had dietary magnesium intakes lower than the recommended dietary allowance (Galan et al., 1997). The deficiency is increased by chronic stress, loud noise exposure or chemotherapy with platinum compounds (Galland, 1991; Mocci et al., 2001, Sahin et al., 2006). Scheibe et al., (1999) were the first to study the correlation between the plasma, perilymph and cerebrospinal magnesium contents in the same animal. Contrary to the blood brain barrier, the blood perilymph barrier is not able to concentrate Mg2+ taken up from plasma. Also, the perilymph Mg2+ concentration correlates well with the plasma level, and a Mg2+ deficiency affects perilymph content. The exact manner by which Mg2+ affects the susceptibility to hearing loss is still unknown, but several mechanisms could be evoked. By its 151

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nature, magnesium is a calcium antagonist and therefore blocks the excessive release of calcium, both in the hair cells and in the cochlear vasculature, limiting cell energy depletion and inducing the vasodilatation of arterioles. Through these mechanisms, magnesium could limit ischemia induced by acoustic trauma. Haupt and Scheibe (2002) demonstrated that cochlear blood flow as well as perilymphatic pO2 levels were significantly increased in noise-traumatized animals that received magnesium supplements. Not only does magnesium combat ischemia, but it is also thought to prevent cell damage caused by hypoxia. Konig et al., (2003) noted a protective effect of magnesium on hypoxia-induced hair cell loss in vitro. In other models, high magnesium concentration has been shown to attenuate the hypoxia/ischemia-induced disruption of the mitochondrial membrane potential, a critical event in triggering cell death (Sharikabad et al., 2001). This mechanism is supported by the observation that an increase in the extracellular magnesium concentration led to a decrease in hypoxia induced apoptosis by maintaining the normal ratio of Bax to Bcl-2 proteins involved in determining the survival of cells or their death (Ravishankar, 2001). Another potential mechanism explaining the Mg2+ efficiency in NIHL or in drug ototoxicity, involves free radical production. Two mechanisms have been suggested (Garcia et al., 1998). It may directly inhibit free radical production, or it may facilitate scavenging of free radicals. Afanasev et al., (1995) showed that Mg2+ inhibits reduced NADP oxidase, an enzyme that produces superoxide radical. However, despite the protective effect of magnesium against oxidative stress, as demonstrated in different models (Sharikabad et al., 2001, Bede et al., 2008), no data are currently available about the cochlea. Lastly, Mg2+ could act on glutamate excitotoxicity, especially in NIHL. Magnesium apparently enhances the survival capability of the cochlear afferents, reducing the effect of glutamate- induced inner hair cell damage (Ehrenberger and

Felix, 1995). Magnesium is able to modulate the opening of Na+/Ca++ channels of the NMDA receptors (Mayer et al., 1984). The blockade of the NMDA receptors by Mg2+ is voltage- dependent, but extracellular Mg2+ behaves as a non-competitive NMDA antagonist, without the side effects presented by the other non- competitive NMDA antagonists. In the hearing process, if Mg2+ is low, an excess of Ca2+ could enter hair cells. In turn, more glutamate would then be produced in response to this Ca2+ influx. Increased glutamate would also greatly increase the activity of the NMDA receptor, which is also operating with low magnesium. With the double insult of high glutamate and low Mg2+, a flood of Ca2+ could go through the NMDA channels into the nerve cell, and the energetic system could be compromised. Conclusion Hearing loss is a significant clinical issue. We now know many of the molecular pathways leading to apoptotic cell death that are triggered by noise and other environmentally mediated traumas such as aminoglycoside antibiotics and chemo- therapeutics. There is increasing evidence for their similarity, since free radical formation and apoptotic cascades have been implicated in all. Interventions can be directed at preventing initial ROS formation, maintaining cochlear blood flow or blocking apoptosis. A large number of therapeutics, including magnesium, have been tested. Magnesium, by its neuroprotective and vasodilatory effects, has the potency to prevent as well as to limit hearing loss, particularly after noise exposure or sudden sensorineural hearing loss. Magnesium therapy at the recommended dosage appears to be safe with few contraindications (such as severe renal failure). In contrast to other therapeutic agents (like corticosteroids), it easily crosses the perilymph blood barrier and reaches the organ of Corti. The majority of studies have shown that magnesium is partly effective. Using magnesium in combination with other agents could improve recovery after hearing loss.

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Henderson D, Bielefeld EC, Harris KC, Hu BH (2006) The role of oxidative stress in noise-induced hearing loss. Ear and Hearing 27:1-19. Hu BH, Henderson D, Nicotera TM (2002) Involvement of apoptosis in progression of cochlear lesion following exposure to intense noise. Hear Res 166:62-71. Hu BH, Henderson D, Nicotera T (2006) Extremely rapid induction of outer hair cell apoptosis in the chinchilla cochlea following exposure to impulse noise. Hear Res 211:16-25. Ising H, Handrock M, Gunther T, Fischer R, Dombrowski M (1982) Increased noise trauma in guinea pigs through magnesium deficiency. Arch Otorhinolaryngol 236:139-46. Joachims Z, Babisch W, Ising H, Guenther T, Handrock M (1983) Dependence of noise-induced hearing loss upon perilymph magnesium concentration. J Acoust Soc Am 74:104-8. Joachims Z, Ising H, Gunther T (1987) Noise-induced hearing loss in humans as a function of serum Mg concentration. Magnes Bull 9:130-1. Konig O, Winter E, Fuchs J, Haupt H, Mazurek B, Weber N, Gross J (2003) Protective effect of magnesium and MK 801 on hypoxia-induced hair cell loss in new-born rat cochlea. Magnes Res 16:98-105. Kopke RD, Liu W, Gabaizadeh R, Jacono A, Feghali J, Spray D (1997) Use of organotypic cultures of Corti's organ to study the protective effects of antioxidant molecules on cisplatin-induced damage of auditory hair cells. Am J Otolaryngol 18:559-71. Le Prell CG, Hughes LF, Miller JM (2007b) Free radical scavengers vitamins A, C, and E plus magnesium reduce noise trauma. Free Radic Biol Med 42:1454-63. Le Prell CG, Yamashita D, Minami SB, Yamasoba T, Miller JM (2007a) Mechanisms of noise-induced hearing loss indicate multiple methods of prevention. Hear Res 226:22-43. Mayer ML, Westbrook GL, Guthrie P (1984) Voltage dependent block by magnesium of NMDA response in spinal cord neurons. Nature 309:261-3. McKeage MJ (1995) Comparative adverse effects of platinium drugs. Drug Safety 13:228-244. Miller JM, Brown JN, Schacht J (2003) 8-iso- prostagladin F(2alpha), a product noise of exposure, reduces inner ear blood flow. Audiol Neurootol 8:207- 21.

Mocci F, Canalis P, Tomasi PA, Casu F, Pettinato S (2001) The effect of noise on serum and urinary magnesium and catecholamines in humans. Occup Med 51:56-61. Nageris B, Ulanovski D, Attias J (2004) Magnesium treatment for sudden hearing loss. Ann Otol Laryngol 113:672-5. Pujol R, Puel JL, Gervais dAldin C, Eybalin M (1990) Physiopathology of the glutamatergic synapses in the cochlea. Acta Oto-Laryngol 113:330-4. Quirk WS, Seidman MD (1995) Cochlear vascular changes in response to loud noise. Am J Otol 16:322-5. Ravishankar S, Ashraf Q, Fritz K, Mishra O, Delivoria- Papadopoulos M (2001) Expression of Bax and Bcl-2 proteins during hypoxia in cerebral cortical neuronal nuclei of newborn piglets: effect of administration of magnesium sulphate. Brain Res 901:23-9. Rybak LP, Ravi R, Somani SM (1995) Mechanism of protection by diethyldithiocarbamate against cisplatin ototoxicity: antioxidant system. Fundam Appl Toxicol 26:293-300. Rybak LP, Withworth CA (2005) Ototoxicity: therapeutic opportunities. Drug Discovery Today 10:1313-21. Sahin AA, Oysu CC, Yilmaz HB, Topak M, Kulekci M, Okar I (2006) Effect of oral magnesium supplementation on cisplatin ototoxicity. J of Otolaryngol 35:112-6. Scheibe F, Haupt H, Ising H (1999) Total magnesium concentrations of perilymph, cerebrospinal fluid and blood in guinea pigs fed different magnesium- containing diets. Eur Arch Otorhinolaryngol 256:215-9. Scheibe F, Haupt H, Ising H (2000b) Preventive effect of magnesium supplement on noise-induced hearing loss in the guinea pig. Eur Arch Otorhinolaryngol 257:10-6. Scheibe F, Haupt H, Ising H, Cherny L (2002) Therapeutic effect of parenteral magnesium on noise- induced hearing loss in guinea pig. Magnes Res 15:27- 36. Scheibe F, Haupt H, Mazurek B, Konig O (2001) Therapeutic effect of magnesium on noise-induced hearing loss. Noise & Health 3:79-84.

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Scheibe F, Haupt H, Vlastos GA (2000a) Preventive magnesium supplement reduces ischemia-induced hearing loss and blood viscosity in the guinea pig. Eur Arch Otorhinolaryngol 257:355-61. Sendowski I, Raffin F, Braillon-Cros A (2006) Therapeutic efficacy of Magnesium after acoustic trauma caused by gunshot noise in guinea pigs. Acta Oto-Laryngol 126:122-9. Sharikabad M, Ostbye K, Lyberg T, Brors O (2001) Effect of extracellular Mg2+ on ROS and Ca2+ accumulation during reoxygenation of rat cardiomyocytes. Am J Phyiol Heart Circ Physiol 280:H344-53. Shibata SB, Raphael Y (2010) Future approaches for inner ear protection and repair. J Commun Disord 43:295-310. Spasov AA, Lobzov MS, Sanzharovskaia NK, Kozhevnikova EV, Kuzubova EA (1999) The effect of polikatan on the ototoxic action of kanamycin. Eksp Klin Farmakol 62:65-6. Thorne RP, Nuttal AL (1987) Laser Doppler measurements of cochlear blood flow during loud sound exposure in guinea pigs. Hear Res 27:1-10. Vormann J, Gnther T (1993) Influence of magnesium on drug-and noise-induced inner ear damage. Animal studies. Schriften Ver Wasser Boden Lufthyg 88:491- 502.

Vormann J, Gnther T (1991) The role of magnesium and zinc in determining vulnerability of the auditory system to salicylate and gentamicin. Arch Compl Env Stud 3:75-82. Walden BE, Henselman LW, Morris ER (2000) The role of magnesium in the susceptibility of soldiers to noise- induced hearing loss. J Acoust Soc Am 108:453-6. World Health Organization (2004) Occupational noise: assessing the burden of disease from work-related hearing impairment at national and local levels. World Health Organization, Series Number 9, Geneva. Williams CJ, Whitehouse JM (1979) Cis-paltinum: a new anticancer agent. Br Med J 23:1689-91. Yamashita D, Jiang H, Schacht J, Miller J (2004) Delayed production of free radicals following noise exposure. Brain Res 1019:201-9. Yang W, Henderson D, Hu BH, Nicotera T (2004) Quantitative analysis of apoptotic and necrotic outer hair cells after exposure to different levels of continuous noise. Hear Res 196:69-76. Zenner H (1986) K+-induced motility and depolarization of cochlear hair cells. Direct evidence for a new pathophysiological mechanism in Menieres disease. Arch Otorhinolaryngol 243:108-11. Zhao F, Manchaiah VK, French D, Price SM (2010) Music exposure and hearing disorders: an overview. Int J Audiol 49:54-64.

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The role of magnesium in pain


Hyo-Seok Na, Jung-Hee Ryu and Sang-Hwan Do*
Department of Anesthesiology and Pain Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea.
* shdo@snu.ac.kr

Abstract Magnesium plays an important role in the prevention of central sensitization and in the attenuation of established pain hypersensitivity, and its main mode of action appears to involve its voltage-gated antagonist action at N-methyl-D-aspartate (NMDA) receptors. Given the putative function of the NMDA receptor in pain transduction, magnesium has been investigated in various clinical conditions associated with acute or chronic pain. The parenteral administration of magnesium, via an intravenous, intrathecal, or epidural route, may reduce pain, and anaesthetic and analgesic requirements during the intra- and post-operative periods. The beneficial effects of magnesium treatment have also been demonstrated in patients suffering from neuropathic pain, such as in those with malignancy-related neurologic symptoms, postherpetic neuralgia, diabetic neuropathy, and chemotherapy-induced peripheral neuropathy. In addition, magnesium therapy has been shown to be effective in alleviating dysmenorrhea, headaches, and acute migraine attacks. Magnesium is playing an evolving role in pain management, but a more thorough understanding of the mechanisms underlying its antinociceptive action and additional clinical studies are required to clarify its role as an analgesic adjuvant.

Introduction The research interest in NMDA receptors has led to an examination of the interactions between NMDA receptors and the induction and maintenance of central sensitization after nociceptive stimuli (Woolf and Thompson, 1991). Ketamine and magnesium are representative NMDA receptor antagonists, and in particular, magnesium can regulate calcium access into cells by antagonizing the NMDA receptor (Paoletti and Neyton, 2007), which has encouraged investigations on the use of magnesium as an analgesic adjuvant. Recent studies have proposed a role for NMDA receptor antagonists in the management of postoperative pain and in other acute and chronic pain conditions. This chapter describes the pharmacologic basis of pain relief by the magnesium ion, and surveys various clinical studies that have examined the antinociceptive effects of magnesium. Mechanism of magnesium as an analgesic adjuvant Although magnesium has no direct analgesic effect, it inhibits calcium ions entering cells by blocking NMDA receptors, which causes an antinociceptive effect. Furthermore, this

antinociceptive effect is related to its prevention of central sensitization caused by peripheral tissue injury (Woolf and Thompson, 1991). Central sensitization is the result of the enhancement of neuronal properties in nociceptive pathways of the central nervous system, and is triggered by repetitive nociceptive afferent inputs, which manifests as a prolonged reduction in the pain threshold. Central sensitization produces pain hypersensitivity, such as wind-up or long-term potentiation of pain, that is, it causes pain even when peripheral stimuli are not intense and continues to cause pain after the initiating stimuli have disappeared (Latremoliere and Woolf, 2009; Woolf, 1983; Woolf and Salter, 2000). Increased intracellular calcium levels seem to play a major role in the initiation of central sensitization (Pockett, 1995; Woolf and Chong, 1993), and the build-up of intracellular calcium is associated with various receptors on postsynaptic neurons of the spinal dorsal horn, such as, NMDA, -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and glutamate receptors (Latremoliere and Woolf, 2009). Of these receptors, NMDA receptor activation has been demonstrated to be essential for initiating and maintaining central sensitization. 157

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The NMDA receptor is a membrane ion channel expressed in the central nervous system. It is a tetramer composed of four different subunits, that is, two NR1 and two NR2 (Dingledine et al., 1999). NMDA receptors regulate the cellular inflows of Na+ and Ca2+, and the outflow of K+. This voltage-dependent ion channel is blocked non-competitively in the resting state by the magnesium ion and by ketamine (phencyclidine site blockade), MK-801, memantine, and others (Felsby et al., 1996; Paoletti and Neyton, 2007) (Fig. 1). On the other hand, the NMDA receptor channel is opened by membrane depolarization induced by the sustained release of glutamate and neuropeptides, which include substance P and calcitonin gene-related peptide (CGRP) (Baranauskas and Nistri, 1998; Mayer et al., 1984). Extracellular magnesium blocks the NMDA receptor in a voltage-dependent manner (Mayer et al., 1984), and thus, can prevent the establishment of central sensitization and abolish existing hypersensitivity. Other noncompetitive or competitive NMDA receptor antagonists, such as MK801 and D-CPP, also prevent and reverse the hyperexcitability of neurons produced by nociceptive afferent inputs (Ma and Woolf, 1995; Woolf and Thompson, 1991). Perioperative pain Many authors have investigated the adjuvant role of magnesium in the context of intra- and post- operative analgesia. Magnesium has been shown to be effective for treating intra- and post- operative pain and for blunting autonomic, somatic, and endocrine reflexes to noxious stimuli (Kara et al., 2002; Koinig et al., 1998; Levaux et al., 2003). Usually, magnesium is administered as an i.v. 30-50 mg/kg bolus of magnesium sulphate as a loading dose, and maintained at 6-20 mg/kg/h by continuous infusion until the end of surgery (Koinig et al., 1998; Ryu et al., 2008; Ryu et al., 2009; Wilder- Smith et al., 1997), or for 4 hours after the initial bolus (Seyhan et al., 2006). Tramer et al., (1996) conducted the first prospective, randomized study on the effect of magnesium on analgesic requirements, pain, comfort, and quality of sleep during the immediate postoperative period. They showed 158

Figure 1. Scheme of the NMDA receptor and its ligand binding sites. The NMDA receptor is composed of four subunits (two NR1 and two NR2). In the extracellular region, there are agonist-binding sites for glutamate and glycine, co-agonists for the efficient opening of the ion channel. The ion channel includes the binding sites for the pore block, one of which is for the Mg2+ and the other is for ketamine, MK-801, memantine, and so on. that magnesium sulphate reduces analgesic requirements and discomfort, and improves quality of sleep during the postoperative period, and that it does not cause any adverse effect at 48 h after surgery. Oguzhan et al., (2008) studied the effect of a magnesium sulphate infusion on postoperative requirements for opioids, intraoperative muscle relaxant, inhalational anaesthetic consumption, and post-operative pain during and after lumbar disc surgery. Their results suggested that intraoperative magnesium administration significantly reduced intraoperative muscle relaxant and opioid requirements, and also reduced postoperative pain and opioid use. When used during a variety of surgeries, magnesium was also found to reduce the need for intraoperative anaesthetics and muscle relaxants and to reduce the amount of morphine required to treat postoperative pain. Ryu et al., (2009) compared remifentanil and magnesium during middle ear surgery in terms of postoperative pain and other complications. In this study, magnesium or remifentanil combined with sevoflurane provided adequate hypotensive anaesthesia, but patients in the magnesium group experienced a more comfortable postoperative course with better analgesia, less

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shivering, and less postoperative nausea and vomiting (PONV). Furthermore, the amount of sevoflurane required to maintain surgical anaesthesia was significantly lower in the magnesium group than in the remifentanil group (Ryu et al., 2009). The use of magnesium as an analgesic adjunct has also been found to be beneficial in patients on total intravenous anaesthesia (TIVA). Significant reductions in intraoperative propofol, atracurium, and postoperative morphine consumption were observed in patients undergoing gynaecological surgery (Seyhan et al., 2006). In another study on gynaecologic patients receiving TIVA, post-operative pain scores, cumulative analgesic consumption, and shivering incidents were significantly lower in patients treated with magnesium, to the extent that the authors concluded magnesium sulphate during TIVA improved the quality of postoperative analgesia (Ryu et al., 2008). Furthermore, Choi et al., (2002) found that intravenous magnesium sulphate reduced propofol infusion requirements during the maintenance of propofol-nitrous oxide anaesthesia in patients undergoing total abdominal hysterectomy. The usefulness of adjunctive magnesium for postoperative pain control has also been examined in the context of regional anaesthesia. Recent studies have suggested that magnesium may play a beneficial role in spinal anaesthesia. For example, magnesium was found to prevent the induction of central sensitization by peripheral nociceptive stimulation at a spinal site of action, by blocking NMDA receptors in a voltage-dependent fashion (Woolf and Thompson, 1991). Utilizing the same mechanism, the addition of small doses of magnesium sulphate to local anaesthetics for spinal anaesthesia enhances the duration of anaesthesia and reduces postoperative analgesic requirements and the incidence of side effects of high doses of local anaesthetics and opioids. In animal studies, the intrathecal co-administration of magnesium sulphate during spinal anaesthesia was found to significantly potentiate morphine analgesia in normal rats and in a surgical model of mechanical allodynia (Kroin et al., 2000). In another experimental model, intrathecal magnesium sulphate produced a state of spinal anaesthesia and general sedation in rats that

lasted for around 1 h, and at 6 h post-injection, animals recovered and showed no evidence of neurotoxicity (Bahar et al., 1996). Clinical studies have also shown that intrathecal magnesium sulphate added to fentanyl prolongs analgesia without increasing side effects during labour analgesia (Buvanendran et al., 2002; Ozalevli et al., 2005). Furthermore, the i.v. infusion of magnesium sulphate during spinal anaesthesia was found to improve postoperative analgesia and reduce the cumulative consumption of analgesics after total hip replacement arthroplasty (Hwang et al., 2010). Postoperative adjunctive i.v. magnesium infusion was also found to increase time to analgesic need and to reduce total analgesia consumption after spinal anaesthesia (Apan et al., 2004). Comparatively little is known of the effect of epidural magnesium sulphate, whereas intrathecal magnesium sulphate has been investigated on many occasions. Caudal epidural anaesthesia with lidocaine plus magnesium sulphate was found to produce analgesia of longer duration than lidocaine plus distilled water in cattle (Dehghani and Bigham, 2009). Arcioni et al., (2007) compared intrathecal, epidural, combined intrathecal and epidural magnesium sulphate, and spinal anaesthesia alone (controls) in patients undergoing orthopaedic surgery to investigate whether intrathecal and/or epidural magnesium sulphate could reduce postoperative analgesic requirements. The results obtained suggested that combined intrathecal and epidural magnesium sulphate significantly reduced postoperative analgesic requirements (Arcioni et al., 2007). However, not all investigations have reported postoperative analgesic effects for magnesium sulphate. For example, perioperative i.v. magnesium infusion was not found to reduce postoperative pain or analgesic consumption in patients undergoing abdominal hysterectomy (Ko et al., 2001) or caesarean delivery (Paech et al., 2006). Furthermore, in a recent report issued by Tramer and Glynn (2007), pretreatment with magnesium sulphate was found to have no effect on postoperative pain or analgesic requirements over the first three postoperative days in patients undergoing ambulatory ilioinguinal hernia repair or varicose vein surgery. However, in this study, a single dose (4 g) of intravenous magnesium 159

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sulphate was used instead of a loading dose plus continuous infusion. Although some debate exists concerning the role of magnesium sulphate as an analgesic adjuvant, the consensus is that magnesium sulphate acts to support a neuromuscular blockade. Magnesium acts as a calcium channel blocker at presynaptic nerve terminals and reduces acetylcholine release at the motor endplate (Fisher, 1999). This diminishes muscle fibre excitability and reduces end plate potential amplitudes, which leads to the potentiation of a neuromuscular blockade by nondepolarizing neuromuscular blockers (Fisher, 1999). Some authors have focused on the direct effect of magnesium on neuromuscular blockade (Fuchs-Buder et al., 1995; Fuchs-Buder and Tassonyi, 1996; Kussman et al., 1997; Ross and Baker, 1996; Telci et al., 2002), whereas others concluded that a perioperative adjuvant magnesium infusion enhances neuromuscular blockade (Lee and Kwon 2009; Oguzhan et al., 2008; Ryu et al., 2008; Ryu et al., 2009; Seyhan et al., 2006). Controlled hypotension is sometimes required during surgery to maintain a bloodless operative field, and investigations on perioperative magnesium infusion for the control of hypotension during middle ear surgery decreased the incidences of postoperative nausea and vomiting (PONV) (Ryu et al., 2008; 2009), which could have been due to lower sevoflurane consumption (Apfel et al., 2002; Ryu et al., 2009) rather than any antiemetic effect of the magnesium. Nevertheless, since PONV is one of the most common and distressing complications after surgery, this effect is interesting, because it could be used to benefit, for example, patients undergoing ambulatory surgery. Perioperative i.v. magnesium infusion has another advantageous effect, in that it decreases the incidence of shivering by up to 70-90% (Ryu et al., 2008; Ryu et al., 2009; Tramer and Glynn, 2007). Shivering is one of the leading causes of postoperative discomfort (Alfonsi, 2001), and increases oxygen consumption (Alfonsi, 2001). Thus, the prevention of shivering is one of the most obvious benefits of magnesium sulphate administration in surgical patients. When mixed with local anaesthetic, magnesium may also show beneficial effects in intravenous 160

regional anaesthesia. In one study, magnesium was added to lidocaine for intravenous regional anaesthesia, and was found to improve quality of anaesthesia and analgesia, specifically, sensory and motor block onset times were shorter and postoperative analgesia was better with magnesium (Turan et al., 2005). However, in this study, recovery after intravenous regional anaesthesia was prolonged in the magnesium group (Turan et al., 2005). In contrast, i.v. magnesium infusion during general anaesthesia has not been found to delay recovery from anaesthesia in most investigations (Lee and Kwon, 2009; Ozcan et al., 2007; Ryu et al., 2008). Another consideration during magnesium administration is that magnesium may cause cardiovascular depression by acting as a calcium channel blocker. The consequent inhibition of catecholamine release lowers plasma epinephrine and norepinephrine concentrations after endotracheal intubation, and therefore reduces hypertensive responses during anaesthesia induction (James et al., 1989). Accordingly, magnesium should be used with caution in hypovolemic patients and in those with limited cardiac capacity. Other pain conditions Numerous clinical studies have found that magnesium has beneficial effects in patients suffering from neuropathic pain, dysmenorrhea, tension headache, acute migraine attack, and others. These effects are considered to be due to blockage of the NMDA receptor, attenuation of central sensitization, and muscle relaxing effects. Neuropathic pain Neuropathic pain is caused by damage to or by the dysfunction of a component of the peripheral or central nervous system (Backonja, 2003). Thus, the causes of neuropathic pain are various and include spinal cord injury, multiple sclerosis, diabetic neuropathy, radiation injury, complications of chemotherapy, a malignancy- related neurologic symptom, amongst others. Its common features include burning, a cold sensation, numbness, itching, and a shooting or abnormal sensation called dysesthesia in a continuous or episodic manner. The mechanism responsible for neuropathic pain remains unknown, but peripheral or central sensitization is a prime candidate (Bridges et al., 2001; Ossipov

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et al., 2000). Neuropathic pain is extremely difficult to treat, and is only partially relieved in some 40-60% of patients. Favoured pharmacologic treatments are antidepressants, anticonvulsants, opioids, and NMDA receptor antagonists (O'Connor and Dworkin, 2009). Magnesium has been shown to have beneficial effects on neuropathic pain in some clinical and animal studies. When magnesium sulphate (bolus doses of 500 mg or 1 g) was given intravenously to patients with uncontrolled neuropathic pain due to cancer infiltration, pain intensity and pain relief scores improved (Crosby et al., 2000). In another study, magnesium chloride (bolus 0.16 mmol/kg over 10 minutes followed by continuous infusion at 0.16 mmol/kg/h) reduced protracted pain and regions of allodynia in patients suffering from peripheral neuropathic pain (Felsby et al., 1996). In addition, in an animal model of neuropathic pain induced hyperalgesia subsided after intraperitoneal magnesium therapy (total 150 or 200 mg/kg for 5 days) (Begon et al., 2000). Postherpetic neuralgia Postherpetic neuralgia (PHN), a type of neuropathic pain, is characterized by chronic persistent pain after an acute herpes zoster attack (Rowbotham and Petersen, 2001). Typically, PHN causes a burning, aching, sharp, and lancinating pain that occurs in a continuous or paroxysmal pattern, and is often accompanied by hyperalgesia and allodynia. Brill et al., (2002) showed that magnesium can relieve hyperalgesia and allodynia in PHN. When they administered i.v. magnesium sulphate at 30 mg/kg over 30 min to patients with intractable PHN, pain was either relieved completely or diminished (Brill et al., 2002). Diabetic neuropathy Diabetes mellitus is the most common etiologic factor of peripheral neuropathy, and diabetic neuropathy is often expressed as numbness, a tingling sensation, and hypersensitivity to pain. These symptoms are limited to the fingers and toes during early disease, and then spread to the proximal extremities to produce a characteristic glove and stocking pattern (Head, 2006). Conventional treatments include antidepressants, anticonvulsants, lidocaine patches, opioid or non- opioids analgesics, and various alternative treatments (Head, 2006). Recently, dietary magnesium supplementation has been at the

focus of attention, and oral doses of magnesium sulphate have been found to be effective at reducing thermal pain thresholds in rats. However, appropriate treatment dosages and durations have not been established (Hasanein et al., 2006). De Leeuw et al., (2004) also demonstrated that long-term magnesium supplementation (300 mg daily for 5 years) can favourably influence the progression of diabetic polyneuropathy. Chemotherapy-induced peripheral neuropathy CIPN is one of the most common side effects in cancer patients administered many chemo- therapeutic agents, such as cisplatin, 5- fluorouracil, vinca alkaloids, taxoids, and etoposide (Head, 2006; Hildebrand, 2006; Wolf et al., 2008). The symptoms and signs of CIPN may resolve completely or partially, but are sometimes irreversible (Kannarkat et al., 2007). Because the pathophysiology of CIPN has not been fully elucidated, various agents have been used to prevent or treat CIPN. Recently, it was reported that a Ca2+/Mg2+ infusion before and after oxaliplatin could prevent the development of CIPN (Gamelin et al., 2004), but the overall benefit of this is uncertain because of the different treatment efficacies of oxaliplatin and Ca2+/Mg2+ infusion combinations (Hochster et al., 2007). Furthermore, oxaliplatin metabolites are chelated by calcium and magnesium, and this could explain the observed neuroprotective effect. However, Bujalska et al., (2009) found that pretreatment with magnesium (30 mg/kg, i.p.) enhanced the analgesic effects of opioids in a vincristine-induced neuropathy model, and re- emphasized the antagonistic effect that magnesium has on NMDA receptors. Dysmenorrhea Dysmenorrhea refers to a gynaecological medical complaint characterized by severe uterine cramps associated with menstruation. Dysmenorrhea may precede or co-occur with menstruation, and causes a dull, nauseating, colicky to sharp or cramping pain. The overproduction of uterine prostaglandins or vasopressin, which stimulates myometrial contraction of the uterus, has been identified as a potential contributory factor (Stromberg et al., 1984; Woolf and Chong, 1993). When dysmenorrhea is severe enough to restrict daily activities, nonsteroidal anti-inflammatory drugs 161

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or oral contraceptive pills are required to reduce uterine contraction, and thus, relieve the pain (Harel, 2008). The benefits and safety of magnesium for the treatment of primary dysmenorrhea have also been reviewed (Bettendorf et al., 2008; Proctor and Murphy 2001), but more research is required to verify its efficacy. Briefly, oral magnesium on the day before menstruation and during the first and second days of the menstrual cycle for six consecutive cycles, was found to have a therapeutic effect on dysmenorrhea (Fontana- Klaiber and Hogg, 1990). However, the optimum treatment regimen has yet to be established and the mechanism responsible has not been determined. Nevertheless, it has been hypothesized that magnesium inhibits the biosynthesis of PGF2-, which alleviates myometrial contraction and muscle relaxation by competing with calcium for entry into myometrial cells through voltage-gated channels (Seifert et al., 1989; Zahradnik and Breckwoldt, 1988). Tension headache Tension headaches are characterized by a diffuse, gradual, and pressure-like aching pain bilaterally over the top sides of the cranium (Altura and Altura, 2001). Its prevalence has been reported to range from around 10% to nearly 90%

(Rasmussen et al., 1991; Robbins and Lipton, 2010; Russell et al., 2006). Although the exact aetiology of tension headaches is unknown, several pathophysiologies have been suggested, such as, muscle tension (Jensen and Rasmussen, 1996; Langemark and Olesen 1987), increased myofascial pain sensitivity (Pfaffenrath et al., 1998), platelet aggregation (Mishima et al., 1997), and others. Furthermore, in one study, it was shown that the magnesium ion levels of platelets were lower in patients with a tension headache (Mishima et al., 1997), and that magnesium supplementation appeared to ameliorate headaches, including the tension headaches (Mauskop et al., 1996; Thomas et al., 1994). Conclusion The postoperative analgesic adjuvant role of magnesium and its use as an analgesic therapy for the treatment of acute or chronic pain have been suggested for decades. Its antinociceptive effect has been suggested to be due to the blocking of NMDA receptors, and thus, the prevention of central sensitization. More consistent and convincing evidence is required before magnesium can be viewed as an effective adjuvant pain treatment.


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The role of magnesium in CNS injury


Naomi L. Cook, Frances Corrigan and Corinna van den Heuvel *
Discipline of Anatomy and Pathology & Adelaide Centre for Neuroscience Research, School of Medical Sciences, The University of Adelaide, Adelaide, South Australia 5005, Australia.
* corinna.vandenheuvel@adelaide.edu.au

Abstract Traumatic injury to the central nervous system (CNS) is a leading cause of morbidity and mortality, and represents a significant public health issue. Despite intensive research, no effective neuroprotective therapy exists, and survivors of CNS injury, including traumatic brain injury (TBI) and spinal cord injury (SCI), can be left with severe disabilities that require long-term rehabilitation. Much of the damage that occurs after TBI and SCI develops over time with the primary injury initiating a secondary injury cascade made up of deleterious biochemical and pathophysiological reactions. This delayed development of secondary injury provides a vital opportunity for therapeutic intervention and considerable effort is currently being directed toward identifying these injury factors and developing interventions that may potentially prevent their actions. Magnesium (Mg2+) decline has been identified as playing a key role in the secondary injury process, in part because of its central role in the regulation of a large number of known injury factors and that its decline is associated with the development of motor and cognitive deficits. Mg2+ administration has been extensively investigated both preclinically in TBI and SCI and clinically as a neuroprotectant in TBI with varied success. This chapter focuses on the role of Mg2+ in TBI and SCI pathophysiology, with particular emphasis on Mg2+ as a potential therapeutic agent.

Introduction A decline in Mg2+ following TBI was first identified using in vivo magnetic resonance spectroscopy (Vink et al., 1988) and further declines have subsequently been reported by numerous laboratories following a variety of insults to the CNS including TBI, SCI, stroke and drug intoxication. It has been observed that a traumatic insult to the CNS results in a decrease in the intracellular free Mg2+ concentration of between 40-60%, while the total tissue Mg2+ falls by between 10- 15%. This Mg2+ decline is confined to the site of injury and the decrease is associated with the development of neurological dysfunction. Treat- ment with Mg2+ has been shown to attenuate the neurological changes experimentally following TBI and SCI, however, clinical studies of Mg2+ as a neuroprotective agent show conflicting results (Dhandapani et al., 2008; Temkin et al., 2007). Traumatic CNS injury Injuries to the CNS are a significant problem, with TBI the leading cause of death and disability in people under the age of 40 years (Fleminger and

Ponsford, 2005). In the USA, approximately 1.5 million people are affected by TBI annually. Of these, 230,000 people are hospitalized and survive, 80,000 people develop a TBI-related disability, and 50,000 people die (Pitknen et al., 2005). In addition, SCI affects around 200,000 people in the USA with 10,000 new cases each year (Anonymous, 2008; Buechner et al., 2000) with the cost of caring for patients with SCI in the US estimated to be $9.73 billion (Weaver et al., 2000). Since no effective treatment is available, survivors of TBI and SCI may be left with debilitating neurological deficits that confer social, financial and emotional burdens and adversely affect their quality of life. The neurological damage resulting from traumatic injuries to the CNS is due to both direct, immediate biomechanical processes (the primary injury) and indirect, delayed (secondary) injury mechanisms (Morales et al., 2005) and the neuronal death in TBI and SCI is likely to involve both necrotic and apoptotic pathways (Liou et al., 2003). The primary event is irreversible and in TBI comprises contusions, lacerations, tissue shearing and stretching of nerve fibres (Gentile and 167

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McIntosh, 1993), whereas the most common primary injuries to the spinal cord result from fracture/dislocation or crush injury to the vertebral column. In contrast, secondary injury consists of a series of complex biochemical changes that are triggered by the primary event and evolve over the minutes to days and even months after the insult (Vink and Van Den Heuvel, 2010). A number of secondary injury factors have been identified, including disruption to the blood-brain barrier (BBB), edema, ischemia, hypertension, inflammation, excito- toxicity, oxidative stress and Mg2+ decline, all of which can be deleterious to neuronal cells (Barone and Kilgore, 2006; Cormio et al., 1997; Gentile and McIntosh 1993; Golding, 2002). Secondary injury factors As stated above, secondary injury involves series of complex biochemical changes that are triggered by the primary event and may continue for days to weeks after the insult (Roth and Farls 2000). To provide an example of the secondary injury cascade, shearing of nerve fibres at the time of insult results in massive ion fluxes across cell membranes, loss of membrane potential and rapid release of neurotransmitters from damaged neurons. This may result in excitotoxicity, which can evoke an inflammatory response, thereby stimulating further processes (such as the changes described above), eventually leading to cell death (Vink and Van Den Heuvel, 2004). Secondary injury is associated with significant morbidity and mortality following traumatic CNS injury (Gentile and McIntosh, 1993), but given that it manifests over time, this provides an opportunity to administer a pharmacological agent to impede or prevent further injury and thus improve outcome. Several injury processes relevant to traumatic CNS injury will now be discussed including diffuse axonal injury (DAI), edema, oxidative stress, mitochondrial dysfunction, inflammation and lastly significant discussion will be given to magnesium decline. Diffuse Axonal Injury DAI is a principal cause of coma following TBI, and significantly contributes to TBI-related morbidity and mortality (Li and Feng, 2009). Classically, DAI was categorized as a primary injury, whereby axons are torn and rupture at the time of insult (Adams et al., 1982). However, more recent 168

evidence suggests that DAI is triggered at the time of injury by inertial forces and may evolve over a period of hours to days, progressing from focal disruptions to axons to impaired axonal transport, axolemmal swelling and, ultimately, axonal disconnection (Bki and Povlishock, 2006; Mazzeo et al., 2009; Stone et al., 2004). DAI is generally diagnosed at post-mortem by immunostaining for -amyloid precursor protein (APP) (Gentleman et al., 1993), although recent imaging advances may allow its detection at an earlier stage (Li and Feng, 2009). Edema Cerebral edema is defined as an abnormal accumulation of fluid within the brain parenchyma, resulting in a volumetric enlarge- ment (swelling) of brain tissue (Heo et al., 2005). Swollen tissues may exert harmful effects by increasing pressure in surrounding tissues, leading to ischemia and additional edema (Simard et al., 2007). Swelling has particularly deleterious consequences in the brain, due to the fact that the volume of the intracranial cavity is fixed (Ayata and Ropper, 2002). The resulting rise in intracranial pressure (ICP) with cerebral edema formation is a potentially lethal process (Kimelberg, 1995). Indeed, cerebral edema plays an important role in the outcome of TBI victims: uncontrolled brain swelling with the accompanying rise in ICP is the leading cause of death in TBI patients (Marmarou et al., 2000). Cerebral edema is classified as cytotoxic and vasogenic, depending upon whether or not BBB permeability is increased (Verlooy and Van Reempts, 2005). The BBB is composed of a monolayer of endothelial cells forming tight junctions that prevent the direct interaction of the peripheral circulation with the CNS (Palmer, 2010). Cytotoxic edema refers to the movement of water, Na+ and Cl- from the extracellular to the intracellular space, which results in cell swelling and predisposes cells to oncotic cell death. However, cytotoxic edema does not involve increases in BBB permeability, nor does it contribute to the actual net increase in brain water (Ayata and Ropper, 2002; Simard et al., 2007). Conversely, vasogenic edema is associated with the degradation of tight junctions between endothelial cells of the BBB (Heo et al., 2005). Consequently, the BBB becomes more permeable to macromolecules and permits the movement of

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fluid from capillaries to the extravascular compartment (Heo et al., 2005; Simard et al., 2007); hence vasogenic edema causes a net increase in brain water content (Ayata and Ropper, 2002). In TBI, the increase in BBB permeability may occur by mechanical injury, autodestructive mediators or both (Unterberg et al., 2004). However, despite the potentially lethal consequences of edema in TBI, there is currently no effective therapy to prevent its formation or progression. Treatments such as mannitol, corticosteroids, barbiturates, induction of hypo- thermia, drainage of the cerebrospinal fluid (CSF) and surgical decompression (Ayata and Ropper, 2002; Clausen and Bullock, 2001) have had limited success in managing rises in ICP (Vink and Van Den Heuvel, 2004). Neurogenic inflammation is a local inflammatory reaction of neurons in response to infection, toxins or trauma, which is characterized by vasodilation, plasma extravasation and edema (Black, 2002; Ro et al., 2005; Turner et al., 2006). Studies from our laboratory (Nimmo et al., 2004; Vink et al., 2003) have shown that neurogenic inflammation may be involved in the formation of vasogenic edema and the functional deficits following diffuse TBI in rats, and that the neuropeptide, substance P (SP), is a potent initiator of neurogenic inflammation. Further research from our laboratory (Donkin et al., 2009) has demonstrated that a SP receptor antagonist inhibits BBB breakdown and edema formation, and significantly improves long-term functional outcome and motor deficits resulting from TBI. These important results may provide a novel pharmacological treatment for TBI. Oxidative Stress Reactive oxygen species (ROS) comprise a range of chemical entities, including hydroxyl radicals, peroxynitrite, superoxide anions, hydrogen peroxide, nitric oxide and singlet oxygen (Chong et al., 2005; Ellis, 2007). ROS are highly reactive molecules, possessing one or more unpaired electrons in their outer orbits (Halliwell, 1992). While some ROS are normal by-products of cellular metabolism (Yu, 1994), their over- production can lead to cell injury and tissue damage via reactions with proteins, lipids and nucleic acids, and cellular activation by ROS induces signalling cascades that lead to rises in intracellular Ca2+ concentrations (Alexandrova

and Bochev, 2005; Massullo et al., 2006). When ROS are generated in excess of endogenous antioxidant mechanisms, the result is oxidative stress (Finkel and Holbrook, 2000). Brain tissue is highly susceptible to damage by oxidative stress for a number of reasons. The brain has a high rate of oxygen metabolism, which increases the likelihood of excess ROS production (Chong et al., 2005). Furthermore, it contains high levels of polyunsaturated fatty acids, which are particularly vulnerable to damage by free radicals (Halliwell, 1992). Critically, the brain has relatively low antioxidant and repair capacities compared with other organs (Vink and Nimmo, 2009). All of these factors render brain tissue extremely sensitive to increases in ROS, and oxidative stress is indeed relevant to TBI. Studies demonstrate that the production of ROS is enhanced following TBI, and this is accompanied by an impairment of antioxidant defences (Ansari et al., 2008; Lima et al., 2008). TBI-induced oxidative stress can lead to cytoskeletal damage, mitochondrial dysfunction and altered signal transduction, which can be deleterious to neuronal and vascular cells (Ansari et al., 2008; Chong et al., 2005). Mitochondrial Dysfunction Mitochondria are organelles that are present in all cells and play a critical role in regulating cellular energy production via the electron transport chain (Bayir and Kagan, 2008; Mazzeo et al., 2009). Consequently, it would be expected that disruption to mitochondrial function would have serious adverse consequences to cells, particularly those with a high-energy demand, such as neurons. Indeed, mitochondrial dysfunction is an important factor in TBI pathophysiology that can lead to energy depletion, free radical release and apoptosis, or programmed cell death (Vink and Nimmo, 2009). In experimenta